Nonmalignant Hematologic Disorders Research Articles

Umbilical cord blood mesenchymal stem cells application in hematopoietic stem cells expansion on nanofiber three-dimensional scaffold.

Umbilical cord blood (UCB) hematopoietic stem cells (HSCs) transplantation (HSCTs) is considered as a therapeutic strategy for malignant and nonmalignant hematologic disorders. Nevertheless, the low number of HSCs obtained from each unit of UCB can be a major challenge for using these cells in adults. In addition, UCB is a rich source of mesenchymal stem cells (MSCs) creating hopes for nonaggressive and painless treatment in tissue engineering compared with bone marrow MSCs. This study was designed to evaluate the effects of UCB-MSCs application in UCB-HSCs expansion on the nanoscaffold that mimics the cell's natural niche. To achieve this goal, after flow cytometry confirmation of isolated HSCs from UCB, they were expanded on three-dimensional (3D) poly-l-lactic acid (PLLA) scaffolds fabricated by electrospinning and two-dimensional (2D)-culture systems, such as (1) HSCs-MSCs culturing on the scaffold, (2) HSCs culturing on the scaffold, (3) HSCs-MSCs culturing on 2D, and (4) HSCs culturing on 2D. After 7 days, real-time polymerase chain reaction (PCR) was performed to evaluate the CXCR4 gene expression in the mentioned groups. Moreover, for the next validation, the number of total HSCs, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay, scanning electron microscopy imaging, and colony-forming unit assay were evaluated as well. The results of the study indicated that UCB-MSCs interaction with HSCs in 3D-culture systems led to the highest expansion of UCB-HSCs on day 7. Flow cytometry results showed the highest purity of HSCs cocultured with MSCs. Real-time PCR showed a significant increase in gene expression of CXCR4 in the mentioned group. The highest viability and clonogenicity were detected in the mentioned group too. Considered together, our results suggest that UCB-HSCs and MSCs coculturing on PLLA scaffold could provide a proper microenvironment that efficiently promotes UCB-HSCs expansion and UCB-MSCs can also be considered as a promising candidate for UCB-HSCTs.

Predictors of Donor/Host (D/H) Lineage Specific Chimerism Trends in Pediatric Patients Following Allogeneic Hematopoietic Cell Transplantation

The outcome of allogeneic HCT is dependent on several pre-transplant variables. We hypothesize that these pre-transplant factors may influence the donor/host chimerism post HCT, which in its turn may affect the outcome. We performed an analysis to study lineage specific D/H chimerism patterns of total blood leukocytes, myeloid cells/neutrophils, B-cells, NK-cells and T-cells. All consecutive patients who underwent an allogeneic HCT between 01/2010 to 06/2015 on the Pediatric BMT Service at MSKCC were reviewed. Chimerism analyses were performed by short tandem repeat (STR) polymorphism analysis at the American Red Cross Blood Services (Philadelphia, PA). Lineage specific donor chimerism post HCT was studied including donor chimerism trend, and factors with potential impact on chimerism including: age, disease, graft source, and T-cell depletion (TCD). Preliminary analyzes performed on this cohort included Wilcoxon Rank Test and cox proportional hazard analyses. 140 patients with a median age of 11.5 years were included. They included 97 patients with hematologic malignancies, 27 patients with non-malignant hematologic disorders and 16 patients with immunologic disorders. Cytoreduction included TBI in 48 patients or chemotherapy only in 98 patients. 105 patients received T-cell depleted grafts, 28 patients unmodified marrow or peripheral blood grafts, and 7 patients unrelated cord blood grafts. No patients received donor leukocyte infusions (DLI). Observational studies revealed that full donor chimerism of myeloid cells, B-cells and NK-cells, but not T-cells occurred early post-transplant. In Wilcoxon Rank Test, there was no difference in the percentage of total donor leukocytes at 3 months vs 12 months post HSCT (n=30), while the median of donor T-cell chimerism was 51% at 3 months and 91% at 12 months post HSCT (p The studies of the impact of different factors on chimerism were performed, including age, disease, and type of graft. Loss of donor total leukocytes chimerism between 3 and 12 months post-transplant was significantly more pronounced for patients This preliminary analysis of lineage specific chimerism post-transplant showed that donor T-cells may take one year to fully recover post-transplant, mostly following T-cell depleted grafts, without intervention. Cord blood grafts were associated with high donor chimerism throughout the post-transplant period. Lastly, factors associated with loss of donor chimerism post-transplant were younger age and non-malignant disorders. More in-depth analyses are being performed.

Innate Immune Determinants of Graft-Versus-Host Disease and Bidirectional Immune Tolerance in Allogeneic Transplantation.

The success of tissue transplantation from a healthy donor to a diseased individual (allo-transplantation) is regulated by the immune systems of both donor and recipient. Developing a state of specific non-reactivity between donor and recipient, while maintaining the salutary effects of immune function in the recipient, is called “immune (transplantation) tolerance”. In the classic early post-transplant period, minimizing bidirectional donor ←→ recipient reactivity requires the administration of immunosuppressive drugs, which have deleterious side effects (severe immunodeficiency, opportunistic infections, and neoplasia, in addition to drug-specific reactions and organ toxicities). Inducing immune tolerance directly through donor and recipient immune cells, particularly via subsets of immune regulatory cells, has helped to significantly reduce side effects associated with multiple immunosuppressive drugs after allo-transplantation. The innate and adaptive arms of the immune system are both implicated in inducing immune tolerance. In the present article, we will review innate immune subset manipulations and their potential applications in hematopoietic stem cell transplantation (HSCT) to cure malignant and non-malignant hematological disorders by inducing long-lasting donor ←→ recipient (bidirectional) immune tolerance and reduced graft-versus-host disease (GVHD). These innate immunotherapeutic strategies to promote long-term immune allo-transplant tolerance include myeloid-derived suppressor cells (MDSCs), regulatory macrophages, tolerogenic dendritic cells (tDCs), Natural Killer (NK) cells, invariant Natural Killer T (iNKT) cells, gamma delta T (γδ-T) cells and mesenchymal stromal cells (MSCs).

Development of a Hematology-Specific Fellowship Curriculum for Advanced Practice Providers Utilizing a Needs-Based Assessment

Background: As our healthcare system faces increasing complexity, it is important for all healthcare providers to practice at the full scope of their training. However, there are limited current options for subspecialty training in hematology for advanced practice providers (APPs), a group of health care providers including both nurse practitioners and physician assistants. We are unaware of any existing postgraduate fellowship for APPs focused on education in both malignant and nonmalignant hematologic disorders. We hope that by creation of the first such fellowship, we can better prepare APP graduates with the subspecialty-specific knowledge required to become highly skilled and competent clinicians in hematology.Methods: Using surveyhero.com, we conducted a web-based needs assessment survey which was distributed to 68 APPs currently working in the hematology and blood & marrow transplant (BMT) teams at three Mayo Clinic sites (Rochester, Scottsdale, and Jacksonville). This survey included questions regarding hematology-specific education the respondents had received in their APP schools, as a way to assess participant's perceptions about the readiness to practice in hematology after completion of APP education, and to identify the learning/training needs that are most important for APPs to practice in hematology.Results: Of 68 APPs, 49 (72%; 34 NPs,15 PAs) completed the survey. 57% of respondents were new APP graduates when hired and 35% had no prior work experience in hematology/BMT. All APPs held a Master's or higher degree (doctorate 31%). Thirty-nine (80%) reported <5% of their APP school curriculum was hematology-focused. The majority (92%) felt that the level of subspecialty training they received in APP schools did not adequately prepare them with the full confidence to practice in hematology. Forty-four (90%) APPs believed subspecialty training could help them to become more competent providers for patients with hematologic disorders. Forty-seven (96%) APPs agreed that being confident and knowledgeable in their practice positively impacted their job satisfaction, and most APPs (84%) felt structured hematology training would attract them to stay within the specialty. The educational subjects that APPs identified as most important included variety of malignant and benign hematologic disorders as well as hematopathology, bone marrow transplant, palliative care, transfusion medicine, infectious disease and pharmacology related to hematology practice. The top three effective learning strategies reported by the APP respondents were active learning from direct patient care, cased based teaching, and educational experience during hospital rounds.Conclusions and Discussions: This needs-based assessment educational project revealed to us the significant gaps in subspecialty training of APP education in hematology and confirmed the necessity of this fellowship. The findings of the survey helped us to optimize the fellowship curriculum in order to meet the learning needs of future APP trainees. Development of the subspecialty fellowship program is a step forward in the future training of APPs. We believe our fellowship has the potential to serve as the postgraduate training model for other institutions nationwide. DisclosuresLacy:Celgene: Research Funding.

HLA-a*0101 Expression Correlates with Increased Risk of Severe Cutaneous Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Acute graft-versus-host disease (aGVHD) remains a substantial cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (alloHSCT). aGVHD most commonly affects the skin, for which there is a high treatment response after both unmodified and ex-vivo CD34+ selected/T-cell depleted (TCD) alloHSCT grafts. We identified a cluster of patients, however, who developed severe refractory cutaneous aGVHD and expressed MHC class I HLA-A*0101 allele. Although the effect of HLA polymorphisms on cutaneous aGVHD is unknown, we hypothesized that HLA-A*0101 expression correlates with worse cutaneous aGVHD after alloHSCT.Methods: We evaluated 831 patients who underwent either an unmodified or TCD allograft at a single institution between 03/2010 and 02/2017. We excluded patients who received <8/8 HLA-matched grafts or cord blood transplant. For each patient with cutaneous aGVHD, we assessed donor-recipient HLA-typing (confirming HLA-A*0101 status), as well as time of onset, grade at onset, and highest overall grade of cutaneous aGVHD.Results: Most of the patients underwent alloHSCT after myeloablative conditioning for the treatment of hematologic malignancies or high-risk non-malignant hematologic disorders. A similar proportion received a TCD or an unmodified allograft. Because all patients had 8/8 HLA-identical donors, both donor and recipient either expressed HLA-A*0101 or did not. HLA-A*0101 was expressed in 206 (25%) patients (98 TCD, 108 unmodified) who had similar demographics to patients lacking HLA-A*0101 (Table 1). At day 180, patients expressing HLA-A*0101 had a higher incidence of grade II-IV cutaneous aGVHD when compared to patients lacking HLA-A*0101 expression in both the TCD (12% vs. 5%, p=0.02) and unmodified (16% vs. 9%, p=0.046) cohorts. Similarly, incidence of severe grade III-IV cutaneous aGVHD was higher in the HLA-A*0101 expressing group when compared to the non-expressing group after TCD (8% vs. 3%, p=0.027) and unmodified (11% vs. 4%, p=0.01) alloHSCT (Fig 1). In a multivariate cause-specific Cox model, HLA-A*0101 expression in the TCD alloHSCT cohort correlated with increased risk of grade III-IV cutaneous aGVHD [HR=2.79 (95% CI: 1.07-7.28), p=0.036] after adjusting for whether the donor was related or unrelated. In the unmodified alloHSCT cohort, HLA-A*0101 expression also correlated with an increased risk of grade III-IV cutaneous aGVHD [HR=2.68 (95% CI: 1.24-5.79), p = 0.012)] after adjusting for relationship status of the donor and the type of conditioning regimen. There was no statistically significant difference in OS or transplant-related mortality between HLA-A*0101 expressing vs. non-expressing patients after TCD or unmodified alloHSCT.Conclusions: Donor/recipient expression of HLA-A*0101 correlates with an increased incidence and severity of cutaneous aGVHD after both TCD and unmodified alloHSCT, including severe grade III-IV cutaneous aGVHD. These findings have potential practical implications in the development of cutaneous aGVHD prophylaxis or early therapeutic strategies targeting the skin in this high-risk population. These findings merit further investigation in a larger patient/donor population. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Peroxisome Proliferator Activated Receptor-Delta Controls Fat Oxidation in Alloreactive T Cells

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia and multiple non-malignant hematologic disorders. However, the routine use of alloHSCT remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues in the skin, gastrointestinal tract, and liver. We have previously shown that GVHD-causing T cells increase fat oxidation compared to both syngeneic and naive T cells. To explore this adaptation mechanistically, we studied the role of the transcription factor Peroxisome Proliferator Activated Receptor delta (PPAR-δ) in alloreactive donor T cells during the initiation of GVHD. By day 7 post-transplant, alloreactive T cells up-regulated PPAR-δ >5-fold compared to pre-transplant naive T cells (p<0.0001, Figure 1A). Furthermore, PPAR-δ was necessary for maximally severe GVHD, as major-MHC mismatched B6xDBA2 F1 mice receiving donor T cells deficient in exon 4 of PPAR-δ (PPAR-δ KO) survived longer than mice receiving wildtype (WT) T cells (p<0.007, Figure 1B).We next investigated the mechanism underlying this observed decrease in GVHD severity. As a transcription factor, PPAR-δ controls expression of multiple genes involved in fat transport and oxidation. To determine its role in alloreactive cells, RNA was collected from CD4 and CD8 T cells on day 7 post-transplant and levels of 8 known PPAR-δ targets quantitated by RT-PCR. These 8 targets were selected from a longer list of genes known to be up-regulated in alloreactive cells. Transcript levels of both carnitine palmitoyl transferase-1a (CPT-1a) and CD36 decreased in PPAR-δ KO CD8 T cells (Figure 2A), with decreases in CD36 protein levels confirmed by immunoblot (Figure 2B). Interestingly, changes in CPT-1a and CD36 did not occur in PPAR-δ KO CD4 T cells. To assess the functional consequence of these changes, day 7 WT versus PPAR-δ KO CD8 T cells were plated with 3H-palmitate and fat oxidation measured ex vivo. Consistent with a decrease in expression of genes involved in fat transport and mitochondrial fat import, fat oxidation decreased by >75% in PPAR-δ KO CD8 cells (Figure 2C). However, despite these decreases, the number of PPAR-δ KO CD8 T cells recovered on day 7 post-transplant was equivalent to WT T cells (Figure 3A, left panel). In contrast, PPAR-δ KO CD4 T cell numbers decreased by 30% on day 7, despite equivalent levels of CD36 and CPT1a (Figure 3A, right panel).Finally, we addressed whether pharmacologic inhibition of PPAR-δ might also effectively mitigate GVHD. Administration of the PPAR-δ inhibitor GSK3787 on days 3-6 post-transplant substantially decreased the number of donor T cell recovered on day 7 (Figure 3B), with PPAR-δ impairment corroborated by a decrease in CPT1a gene transcription. However, instead of improving recipient health, GSK3787 treatment instead worsened weight loss and increased rates of post-transplant morbidity and mortality. From these data, we conclude that PPAR-δ is necessary in alloreactive T cells to cause maximally severe GVHD and that mechanistically, an absence of PPAR-δ impairs fat oxidation in CD8 T cells without impacting CD8 T cell numbers. In contrast, PPAR-δ deficiency decreases the number of CD4 T cells post-transplant, but does so without impacting CPT1a or CD36 levels, highlighting clear differences in metabolic reprogramming between CD4 and CD8 alloreactive cells. Finally, our data suggest that systemic inhibition of PPAR-δ post-transplant is not feasible given a sharp increase in toxicity. Future work will elucidate the mechanism of PPAR-δ in CD4 T cells, define the additional metabolic adaptations of CD8 cells which lack PPAR-δ, and determine if similar changes occur in human T cells. Together, these studies will test whether cellular inhibition of PPAR-δ represents a clinically-relevant, future therapy for GVHD. DisclosuresNo relevant conflicts of interest to declare.

CD3+/CD19+ Depleted Matched and Mismatched Unrelated Donor Hematopoietic Stem Cell Transplant with Targeted T Cell Addback Is Associated with Excellent Outcomes in Pediatric Patients with Nonmalignant Hematologic Disorders

Unrelated donor hematopoietic stem cell transplantation (HSCT) is increasingly being used to cure nonmalignant hematologic diseases (NMHD) in patients who lack HLA matched related donors. Both graft rejection and graft-versus-host disease (GVHD) remain major barriers to safe and effective transplant for these patients requiring unrelated donors. Partial T cell depletion combined with peripheral stem cell transplantation (pTCD-PSCT) has the potential advantages of providing a high stem cell dose to facilitate rapid engraftment, maintaining cells that may facilitate engraftment, and decreasing GVHD risk compared with T cell–replete HSCT. Here, we report a single-institution, retrospective experience of unrelated donor pTCD-PSCT for pediatric patients with NMHD. From 2014 to 2017, 12 pediatric patients with transfusion-dependent NMHD underwent matched unrelated donor (MUD) or mismatched unrelated donor (MMUD) pTCD HSCT in our center using disease-specific conditioning. Donor PSCs underwent CD3+ T cell and CD19+ B cell depletion using CliniMACS, followed by a targeted addback of 1 × 105 CD3+ T cells/kg to the graft before infusion. All 12 patients demonstrated rapid trilinear engraftment. At a median follow-up of 740days (range, 279 to 1466), all patients were alive with over 92% total peripheral blood donor chimerism and without transfusion dependence or recurrence of their underlying hematologic disease. Immune reconstitution was rapid and comparable with T cell–replete HSCT. No patients developed severe acute GVHD (grades III to IV) or chronic extensive GVHD, and all patients had discontinued systemic immune suppression. Viral reactivations were common, but no patient developed symptoms of life-threatening infectious disease. Our data indicate that MUD and MMUD pTCD-PSCTs are safe and effective approaches that enable rapid engraftment and immune reconstitution, prevent severe GVHD, and expand availability of HSCT to any patients with NMHD who have closely MUDs.

Utility and Safety of Liver Biopsy in Patients with Undetermined Liver Blood Test Anomalies after Allogeneic Hematopoietic Stem Cell Transplantation: A Monocentric Retrospective Cohort Study

Liver blood test anomalies are common after allogeneic hematopoietic stem cell transplantation (allo-HSCT), but their cause often remains difficult to identify. Our objective was to evaluate the safety and utility of liver biopsies in patients who underwent allo-HSCT. In a retrospective single-center cohort study, we reviewed all cases of patients who underwent liver biopsy between June 2005 and July 2017. During this period, 54 biopsies were performed in 45 patients, in which 38 patients underwent allo-HSCT for malignant and 7 for nonmalignant hematological disorders. Median time between allo-HSCT and liver biopsy was 213 days. Seven biopsies were percutaneous, and 47 were transjugular. No adverse event related to the biopsy procedure occurred; 94.5% biopsies (51 of 54) led to a histological diagnosis. Cholestatic graft-versus-host disease was histologically demonstrated in 16 biopsies (30%); hepatitis-like graft-versus-host disease in 9 biopsies (17%); nonalcoholic steatohepatitis in 6 biopsies (9%); regenerative nodular hyperplasia in 4 biopsies (5%); and drug-induced liver injury, sinusoidal obstruction syndrome, and viral hepatitis each in 3 biopsies (5%). Association between clinical, laboratory, imaging and pathological features was poor. Only 34% of physicians' prebiopsy hypotheses were confirmed by pathological findings. Patient management was influenced by liver biopsy results in 65% of cases, allowing us to identify a new diagnosis (n = 13), rule out a differential diagnosis (n = 14), or confirm the main hypothesis (n = 6). In conclusion, liver biopsy is a safe and useful technique to investigate liver blood test anomalies following allo-HSCT.

Animal Models for Preclinical Development of Allogeneic Hematopoietic Cell Transplantation.

Since its inception in the 1950s, hematopoietic cell transplantation (HCT) has become a highly effective clinical treatment for malignant and nonmalignant hematological disorders. This milestone in cancer therapy was only possible through decades of intensive research using murine and canine animal models that overcame what appeared in the early days to be insurmountable obstacles. Conditioning protocols for tumor ablation and immunosuppression of the recipient using irradiation and chemotherapeutic drugs were developed in mouse and dog models as well as postgrafting immunosuppression methods essential for dependable donor cell engraftment. The random-bred canine was particularly important in defining the role of histocompatibility barriers and the development of the nonmyeloablative transplantation procedure, making HCT available to elderly patients with comorbidities. Two complications limit the success of HCT: disease relapse and graft versus host disease. Studies in both mice and dogs have made significant progress toward reducing and to some degree eliminating patient morbidity and mortality associated with both disease relapse and graft versus host disease. However, more investigation is needed to make HCT more effective, safer, and available as a treatment modality for other non-life-threatening diseases such as autoimmune disorders. Here, we focus our review on the contributions made by both the murine and canine models for the successful past and future development of HCT.

Clinical Significance of Lymphoid Enhancer-Binding Factor 1 (LEF-1) Expression in Acute Myeloid Leukemia

Background: Lymphoid enhancer-binding factor-1 (LEF1) is a member of the LEF/T-cell factor family of transcription factors and a key mediator of the Wingless-type (Wnt) pathway. It mediates Wnt signals through recruiting β-catenin and its co-activators to Wnt response elements of target genes. It plays crucial roles in normal hematopoiesis, not only in the development of B and T lymphocytes but also in granulopoiesis. Objectives: The aim of this study was to evaluate of LEF1 expression levels in patients with AML and correlate this expression with clinical data. Methodology: The present study was conducted on 30 de novo adult AML patients and 10 age and sex-matched controls with non-malignant hematological disorders (e.g.: hypersplenism, megaloblastic anemia, immune thrombocytopenic purpura) who attended Hematology/Oncology unit of Ain-Shams University Hospitals during the period from May 2017 till January 2018, after taking the approval of the Scientific and Ethical Committee of Ain- Shams University. LEF1 expression levels were measured by quantitative real-time PCR. Results: The present study showed that there was highly statistically significant association between patients and control group in LEF1 expression level. There was positive significant correlation between LEF1 expression level and Hb level and PLT count. While negative significant correlation was found with age, TLC count and peripheral blood blast %. No correlation was found with bone marrow blasts %. There was statistically significant difference between LEF1 expression level and both hepatosplenomegaly. No statistically significant difference was found between LEF1 expression level and Sex or lymphadenopathy. Conclusion: Our study has shown that Lymphoid Enhancer-Binding Factor 1 (LEF-1) is over expressed in AML patients. LEF1 expression might be involved in the process of disease progression, and possibly can serve as a molecular parameter for risk assessment and/or monitoring of treatment in AML patients. Thus, molecular assessment of LEF1 expression at diagnosis may be of value to add to the prognostic work of AML patients.

A Low Incidence of Cytomegalo Virus Infection Following Allogeneic Hematopoietic Stem Cell Transplantation Despite a High Seroprevalence.

Cytomegalovirus (CMV) infection remains an important cause of morbidity and mortality following allogeneic stem cell transplantation (SCT). We wanted to study if the high sero-prevalence seen in our population translated into a high incidence of CMV infection following SCT. This is a retrospectiveanalysis of patients who underwent allogeneic SCT between January 2008 and December 2012 at our centre. 475 patients underwent allogeneic SCT for malignant (46.5%) and non-malignant (53.5%) haematological disorders. 463 (97.4%) SCT recipients and 403 (84.8%) SCT donors were IgG seropositive for CMV. CMV reactivation within 100days post SCT was seen in 174 (36.6%) at a median of 41days (range 10-100) post SCT. Ganciclovir was used in 166 patients (95.4%) for a mean duration of 16days (range 5-32). 157 patients (90%) responded to therapy. Sixty-six patients (42.3%) had secondary reactivation of the virus. Use of a male donor (p = 0.000), donor and recipient age > 15 (p = 0.005 and 0.000), unrelated donor (p = 0.000), degree of HLA mismatch (p = 0.000), occurrence of acute GVHD (p = 0.000) and steroid refractory acute GVHD (p = 0.026) were identified as risk factors for CMV reactivation while early neutrophil recovery (< 15days) was found to be protective (p = 0.004). On multivariate analysis, male donor (p = 0.042), degree of HLA mismatch (p = 0.006), the occurrence of acute GVHD (p = 0.000) and steroid refractory acute GVHD (p = 0.031) continued to remain significant. 5-year overall survival was significantly better in patients without CMV reactivation compared to those who developed reactivation of CMV (68.9 ± 3.7 vs 58.2 ± 4.9% p = 0.004). The incidence of CMV infection does not seem to be higher despite a high sero-prevalence of CMV. However, patients who developed CMV infection post SCT had inferior outcomes.

Late Effects Screening Guidelines after Hematopoietic Cell Transplantation (HCT) for Hemoglobinopathy: Consensus Statement from the Second Pediatric Blood and Marrow Transplant Consortium International Conference on Late Effects after Pediatric HCT

Allogeneic hematopoietic cell transplantation (HCT) can halt organ damage and eliminate symptoms in hemoglobin disorders, including sickle cell disease (SCD) and thalassemia major. Managing the residual manifestations of pre-HCT disease complications and the long-term effects of HCT requires systematic monitoring, follow-up and intervention when indicated. Late complications vary with age and disease status at HCT and with transplant variables such as preparative regimen, donor source and compatibility, and immune reconstitution. An international consensus conference sponsored by the Pediatric Blood and Marrow Transplant Consortium in May 2016 entitled “Late Effects Screening and Recommendations Following HCT for Immune Deficiency and Nonmalignant Hematologic Disorders” focused on follow-up after HCT for hemoglobinopathy. An earlier publication from experts who participated in this session described the pathophysiology and spectrum of complications that HCT recipients experience after HCT for SCD and thalassemia major. This companion publication summarizes the consensus reached by this group of experts about long-term follow-up guidelines after HCT for hemoglobinopathy. In addition, these guidelines might also be included in studies of novel curative therapies such as autologous HCT after hematopoietic progenitor stem cell gene modification.

A human bone marrow mesodermal-derived cell population with hemogenic potential.

The presence, within the human bone marrow, of cells with both endothelial and hemogenic potential has been controversial. Herein, we identify, within the human fetal bone marrow, prior to establishment of hematopoiesis, a unique APLNR+, Stro-1+ cell population, coexpressing markers of early mesodermal precursors and/or hemogenic endothelium. In adult marrow, cells expressing similar markers are also found, but at very low frequency. These adult-derived cells can be extensively culture expanded in vitro without loss of potential, they preserve a biased hemogenic transcriptional profile, and, upon in vitro induction with OCT4, assume a hematopoietic phenotype. In vivo, these cells, upon transplantation into a fetal microenvironment, contribute to the vasculature, and generate hematopoietic cells that provide multilineage repopulation upon serial transplantation. The identification of this human somatic cell population provides novel insights into human ontogenetic hematovascular potential, which could lead to a better understanding of, and new target therapies for, malignant and nonmalignant hematologic disorders.

Protein dysregulation in graft versus host disease.

Allogeneic hematopoietic stem cell transplantation is a well-established treatment for many malignant and non-malignant hematological disorders. As a frequent complication in up to 50% of all patients, graft-versus-host disease is still the main cause for morbidity and non-relapse mortality. Diagnosis is usually done clinically, even though confirmation by pathology is often used to support the clinical findings. Effective treatment requires intensified immunosuppression as early as possible. Although several promising biomarkers have been proposed for an early diagnosis, no internationally-recognized consensus has yet been established. Protein-based biomarkers represent an interesting tool since they have been recently reported to be an important regulator of various cells, including immune cells such as T cells. Therefore, we assume that protein dysregulation is important in the pathogenesis of acute graft versus host disease and their detection might be an possibility in the early diagnosis and monitoring. In this review, we aim to summarize the previous reports of protein biomarkers, focusing on the pathogenesis of the disease and possible implications in diagnostic approaches.

Gene Therapy: Where We Started and Where the Field Is Going

In the more than 20 years since the first successful gene transfer was performed in a human subject (Blaese, R.M. et al. Science 1995; 270, 475-480), the promise of gene therapy has been at once tantalizingly close and just out of reach. Today, gene-modified therapies are being used to treat a variety of diseases, and direct manipulation of the human genome for therapeutic effect is becoming a reality. While many advances in gene-based therapeutics have come in the oncology space, non-malignant hematologic and primary immune disorders remain important targets for these approaches. The ability to manipulate hematopoietic stem cells (HSCs) ex vivo and return the modified HSCs to the patient offers many potential advantages over allogeneic HSC transplantation. Additionally, the potential to introduce new genes with therapeutic potential, or to modify genes to modulate protein expression, may open new avenues for transformative therapies for genetic diseases. The progress of gene therapy for hemoglobinopathies - from the γ-retroviral vector technology that established the field, to the accumulating clinical experience with lentiviral vector-based gene therapy and the potential for gene editing-based approaches to address these diseases - provides insight into the development of genetic therapeutics. Transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) result from pathogenic mutations in the β-globin gene, HBB . Addition of functional HBB genes into autologous hematopoietic stem cells has the potential to offer the long-term therapeutic benefits of allogeneic HSC transplantation without the complications of graft vs host disease. The first human proof of concept of this approach came in a study of the HPV569 lentiviral vector coding for therapeutic β-globin, which was successfully introduced into the HSCs of patients with TDT and resulted in sustained clinical benefit in some patients (Cavazzana-Calvo et al., Nature 2010; 467(7313):318-22). Beyond restoring normal β-globin production, studies with HPV569 and its improved variant BB305, have shown that it is possible to drive expression of a β-globin variant with a point mutation, T87Q, designed to mimic the anti-sickling effect of γ-globin. This innovation may have important implications for patients with SCD, where introduction of normal β-globin may be insufficient to ameliorate the red blood cell sickling and polymerization that can cause painful and damaging vaso-occlusive crises. It has taken more than 20 years for HSC gene addition to reach safety and efficacy thresholds that may allow it to be used routinely for patients with hemoglobinopathies, but this approach is now nearing maturity. Important refinements in LVV architecture and advances in HBB gene cassette design have yielded promising results in multiple clinical studies of TDT and SCD (e.g. Ribeil J.A. et al., N Engl J Med 2017; 376:848-855). There is also significant excitement about the potential for gene editing approaches to address the hemoglobinopathies even as these technologies are just beginning to transition from lab to clinic. Critical questions of both efficacy and safety remain regarding the path forward for nuclease-based editing technologies such as CRISPR, ZFN, and megaTALs. Key lessons from the development of clinical gene addition therapies in the hemoglobinopathies may help chart the path forward for gene editing technologies. Disclosures Gregory: bluebirdbio: Employment; Merck KGaA: Membership on an entity's Board of Directors or advisory committees.

The Effect of Mesenchymal Stem Cell-Derived Extracellular Vesicles on Hematopoietic Stem Cells Fate.

Hematopoietic stem cells (HSCs) are multipotent stem cells, with self-renewal ability as well as ability to generate all blood cells. Mesenchymal stem cells (MSCs) are multipotent stem cells, with self-renewal ability, and capable of differentiating into a variety of cell types. MSCs have supporting effects on hematopoiesis; through direct intercellular communications as well as secreting cytokines, chemokines, and extracellular vesicles (EVs). Recent investigations demonstrated that some biological functions and effects of MSCs are mediated by their EVs. MSC-EVs are the cell membrane and endosomal membrane compartments, which are important mediators in the intercellular communications. MSC-EVs contain some of the molecules such as proteins, mRNA, siRNA, and miRNA from their parental cells. MSC-EVs are able to inhibit tumor, repair damaged tissue, and modulate immune system responses. MSC-EVs compared to their parental cells, may have the specific safety advantages such as the lower potential to trigger immune system responses and limited side effects. Recently some studies demonstrated the effect of MSC-EVs on the expansion, differentiation, and clinical applications of HSCs such as improvement of hematopoietic stem cell transplantation (HSCT) and inhibition of graft versus host disease (GVHD). HSCT may be the only therapeutic choice for patients who suffer from malignant and non-malignant hematological disorders. However, there are several severe side effects such GVHD that restricts the successfulness of HSCT. In this review, we will discuss the most important effects of MSCs and MSC-EVs on the improvement of HSCT, inhibition and treatment of GVHD, as well as, on the expansion of HSCs.

Dissecting Regulatory Mechanisms Using Mouse Fetal Liver-Derived Erythroid Cells.

Multipotent hematopoietic stem cells differentiate into an ensemble of committed progenitor cells that produce the diverse blood cells essential for life. Physiological mechanisms governing hematopoiesis, and mechanistic aberrations underlying non-malignant and malignant hematologic disorders, are often very similar in mouse and man. Thus, mouse models provide powerful systems for unraveling mechanisms that control hematopoietic stem/progenitor cell (HSPC) function in their resident microenvironments in vivo. Ex vivo systems, involving the culture of HSPCs generated in vivo, allow one to dissociate microenvironment-based and cell intrinsic mechanisms, and therefore have considerable utility. Dissecting mechanisms controlling cellular proliferation and differentiation is facilitated by the use of primary cells, since mutations and chromosome aberrations in immortalized and cancer cell lines corrupt normal mechanisms. Primary erythroid precursor cells can be expanded or differentiated in culture to yield large numbers of progeny at discrete maturation stages. We described a robust method for isolation, culture, and analysis of primary mouse erythroid precursor cells and their progeny.

T-Cell Manipulation Strategies to Prevent Graft-Versus-Host Disease in Haploidentical Stem Cell Transplantation.

Allogeneic haematopoietic stem cell transplantation (HSCT) from an human leukocyte antigen (HLA)-identical donor can be curative for eligible patients with non-malignant and malignant haematological disorders. HSCT from alternative donor sources, such as HLA-mismatched haploidentical donors, is increasingly considered as a viable therapeutic option for patients lacking HLA-matched donors. Initial attempts at haploidentical HSCT were associated with vigorous bidirectional alloreactivity, leading to unacceptably high rates of graft rejection and graft-versus-host disease (GVHD). More recently, new approaches for mitigating harmful T-cell alloreactivity that mediates GVHD, while preserving the function of tumour-reactive natural killer (NK) cells and γδ T cells, have led to markedly improved clinical outcomes, and are successfully being implemented in the clinic. This article will provide an update on in vitro strategies and in vivo approaches aimed at preventing GVHD by selectively manipulating key components of the adaptive immune response, such as T-cell receptor (TCR)-αβ T cells and CD45RA-expressing naive T cells.

Prevalence, Risk Factors, and Outcomes of Bronchiolitis Obliterans After Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is a therapeutic approach for malignant and nonmalignant hematological disorders. Survival rates increased after allogeneic HSCT, and therefore, late transplant-related complications have become more frequent. We retrospectively analyzed the incidence, risk factors, and outcome of obliterative bronchiolitis. Between January 2000 and November 2012, 89 transplanted pediatric patients were retrospectively evaluated. The diagnosis of bronchiolitis obliterans was made according to the NIH consensus criteria for chronic graft-versus-host disease (cGVHD). Univariate and multivariate analysis was conducted on the following risk factors: age at HSCT, sex, high risk, type of donor and type of diagnosis, conditioning regimen, total body irradiation, busulfan-based regimen, acute and cGVHD, donor sources, ABO blood group incompatibility, Rh incompatibility, human leukocyte antigen (HLA), and cytomegalovirus (CMV) reactivation. Seven patients met the diagnostic criteria. The prevalence was 7.8%. Pulmonary function tests (PFTs) performed in all bronchiolitis obliterans (BO) patients did not present any abnormality. PFT variables decreased significantly. Four patients presented with moderate forms of obliterative bronchiolitis, 1 patient was identified as having mild form, and 2 patients were categorized with severe obliterative bronchiolitis. cGVHD, both in hepatic and cutaneous form, was demonstrated in the present study to be a major risk factor. The higher incidence of bronchiolitis obliterans in children with hepatic and cutaneous GVHD could be potentially interpreted on the basis that cGVHD is an immunoregulatory disorder that involves almost any organ of the body, and therefore, BO could be described as the pulmonary manifestation of a systemic disease.

Bone Marrow Homing and Engraftment Defects of Human Hematopoietic Stem and Progenitor Cells.

Homing of hematopoietic stem cells (HSC) to their microenvironment niches in the bone marrow is a complex process with a critical role in repopulation of the bone marrow after transplantation. This active process allows for migration of HSC from peripheral blood and their successful anchoring in bone marrow before proliferation. The process of engraftment starts with the onset of proliferation and must, therefore, be functionally dissociated from the former process. In this overview, we analyze the characteristics of stem cells (SCs) with particular emphasis on their plasticity and ability to find their way home to the bone marrow. We also address the problem of graft failure which remains a significant contributor to morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Within this context, we discuss non-malignant and malignant hematological disorders treated with reduced-intensity conditioning regimens or grafts from human leukocyte antigen (HLA)-mismatched donors.