Human Leukocyte Antigen-matched Donor Research Articles

Ocular graft host disease after allogeneic stem cell transplant: unique risk factors and outcomes

Objective The aim of this study was to formulate a retrospective study investigating the ocular findings and associated risk factors in long-term survivors after allogenic-hematopoietic stem cell transplantation (HSCT) in Saudi Arabia. Patients and methods This observational study was conducted on patients who underwent HSCT from 1996 to 2016 at King Faisal Specialist Hospital and Research Center in Riyadh, Saudi Arabia. The study included 72 patients who were human leukocyte antigen-matched donors with identical matched sibling donors. Peripheral blood was the source of stem cells in 61 patients. The ophthalmological examination included visual acuity (VA) testing, refraction, tear break-up time, Schirmer’s test, slit-lamp biomicroscopy, and dilated ophthalmoscopy. Chronic ocular graft-versus-host disease (GVHD) inclusion was based on strict selection criteria. The patients identified with mild to moderate ocular GVHD had less than or equal to three corneal staining, 13–32 points ocular surface disease index (OSDI), and Schirmer’s test of 6–15 mm were classified as group 1, while those with severe ocular GVHD defined as having more than four corneal stainings, more than or equal to 33 points OSDI, and less than or equal to 5 mm Schirmer’s test were classified as group 2. Impairment of VA was classified as mild (20/22–20/50), moderate (20/70–>20/200), or severe (≤20/200) impairment. Results Chronic GVHD was diagnosed in 394 patients within the first 5 years after transplantation. Ocular GVHD was detected in 81 (20.56%) patients. They were classified into group 1 (65 patients, 80.25%) with mild to moderate ocular GVHD and group 2 (16 patients, 19.75%) with severe ocular GVHD. All patients had OSDI ranging from more than or equal to 13 to more than or equal to 33. Retinal changes were detected in five patients, optic disc changes in two patients, steroid-induced proliferative diabetic retinopathy in three patients, and old branch retinal vein occlusion in one patient. All patients with identical matched sibling donor showed normal or mild to moderate impaired VA except for two patients. All patients responded to topical eye drops except for nine who required surgical procedures. Normal or mild impaired VA was reported in 59 patients with statistical significance (P=0.0053), while 42 male patients versus 29 females had normal or mild impaired VA with a statistically significant difference (P=0.0179). Conclusion Ocular GVHD is a complex disease in our unique population. Every patient before allogenic HSCT be referred to a cornea specialist to evaluate the baseline parameters for the pre-HSCT diagnosis of dry eye disease.

Cost-Effectiveness of Reduced-Intensity Allogeneic Hematopoietic Cell Transplantation for Older Patients With High-Risk Myelodysplastic Syndrome: Analysis of BMT CTN 1102.

BMT CTN 1102 was a phase III trial comparing reduced-intensity allogeneic hematopoietic cell transplantation (RIC alloHCT) to standard of care for persons with intermediate- or high-risk myelodysplastic syndrome (MDS). We report results of a cost-effectiveness analysis conducted alongside the clinical trial. Three hundred eighty-four patients received HCT (n = 260) or standard of care (n = 124) according to availability of a human leukocyte antigen-matched donor. Cost-effectiveness was calculated from US commercial and Medicare perspectives over a 20-year time horizon. Health care utilization and costs were estimated using propensity score-matched cohorts of HCT recipients in the OptumLabs Data Warehouse (age 50-64 years) and Medicare (age 65 years and older). EuroQol 5 Dimension (EQ-5D) surveys of trial participants were used to derive health state utilities. Extrapolated 20-year overall survival for those age 50-64 years was 29% for HCT (n = 105) versus 13% for usual care (n = 44) and 31% for HCT (n = 155) versus 12% for non-HCT (n = 80) for those age 65 years and older. HCT was more effective (+2.36 quality-adjusted life-years [QALYs] for age 50-64 years and +2.92 QALYs for age 65 years and older) and more costly (+$452,242 in US dollars (USD) for age 50-64 years and +$233,214 USD for age 65 years and older) than usual care, with incremental cost-effectiveness ratios of $191,487 (USD)/QALY and $79,834 (USD)/QALY, respectively. For persons age 50-64 years, there was a 29% chance that HCT was cost-effective using a willingness-to-pay (WTP) threshold of $150K (USD)/QALY and 51% at a $200K (USD)/QALY. For persons age 65 years and older, the probability was 100% at a WTP >$150K (USD)/QALY. Among patients age 65 years and older with high-risk MDS, RIC HCT is a high-value strategy. For those age 50-64 years, HCT is a lower-value strategy but has similar cost-effectiveness to other therapies commonly used in oncology.

Clinical Effects of Tacrolimus Blood Concentrations Early after Allogeneic Hematopoietic Stem Cell Transplantation

Tacrolimus (TAC) plus short-term methotrexate (stMTX) is used for graft-versus-host disease (GVHD) prophylaxis after allogeneic hematopoietic stem cell transplantation (allo-HSCT). TAC blood concentrations are frequently adjusted to enhance the graft-versus-leukemia/lymphoma effect or attenuate severe GVHD. Limited information is available on the clinical impact of these adjustments and the optimal time to perform them in order to achieve good clinical outcomes. We retrospectively analyzed 211 patients who underwent allo-HSCT at our institutes. Higher TAC concentrations in week 3 correlated with a significantly higher cumulative incidence of relapse (CIR) (p=0.03) and lower non-relapse mortality (p=0.04). The clinical impact of high TAC concentrations in week 3 on CIR was detected in the refined disease risk index: low/intermediate (p=0.04) and high (p<0.01), and conditioning regimens other than cyclophosphamide/total body irradiation and busulfan/cyclophosphamide (p=0.07). Higher TAC concentrations in week 1 correlated with a lower grade 2-4 acute GVHD rate (p=0.01). Higher TAC concentrations in weeks 2 and 3 correlated with slightly lower (p=0.05) and significantly lower (p=0.02) grade 3-4 acute GVHD rates, respectively. Higher TAC concentrations in weeks 1 and 3 were beneficial for severe acute GVHD in patients with a human leukocyte antigen-matched donor (p=0.03 and p<0.01, respectively), not treated with anti-thymocyte globulin (p=0.02 and p=0.02, respectively), and receiving three stMTX doses (p=0.03 and p=0.02, respectively). The clinical impact of TAC concentrations varied according to patient characteristics, including disease malignancy, conditioning regimens, donor sources, and GVHD prophylaxis. These results suggest that TAC management needs to be based on patient profiles.

Prime Editing Efficiently and Precisely Corrects Causative Mutation in Chronic Granulomatous Disease, Restoring Myeloid Function: Toward Development of a Prime Edited Autologous Hematopoietic Stem Cell Therapy

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by a lack of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in phagocytic myeloid cells such as neutrophils. NADPH oxidase produces reactive oxygen species (ROS) that directly kill bacteria and fungi to control infection. CGD patients lack this ability to control infection, which results in chronic and severe infections, inflammation, autoimmunity, poor quality of life, and reduced lifespan. Current standard care for CGD is antimicrobial prophylaxis and aggressive management of infection. For patients with a suitable human leukocyte antigen (HLA) matched donor, an allogeneic hematopoietic stem cell (HSC) transplant (allo-HSCT) offers a curative approach. However, many patients do not have access to HLA matched donors and allo-HSCT also carries a risk of GvHD and graft rejection. P47phox-deficient CGD, the most common autosomal recessive CGD, is caused by mutations in the NCF1 gene, which encodes the p47phox protein, a subunit of the NADPH oxidase complex. Approximately 80% of p47phox-deficient CGD patients carry the same homozygous mutation, a 2 nucleotide GT deletion (delGT) in exon 2 of NCF1 which is correctable using Prime Editing. Transplantation of Prime Edited patient autologous CD34 + cells, in which the delGT mutation has been corrected with Prime Editing, may provide a curative treatment for p47phox-deficient CGD patients. To evaluate whether correction of the delGT NCF1 mutation in CGD patient CD34 + cells restores p47phox protein expression and NADPH oxidase activity in myeloid progeny, Prime Editors were designed to correct this mutation precisely and efficiently. The Prime Editors were delivered to CD34 + cells from two p47phox-deficient CGD patients homozygous for delGT NCF1. In both studies, ≧75% of Prime Edited patient CD34 + cells carried at least 1 corrected allele as determined by ddPCR and DNA sequencing analyses. Myeloid progeny differentiated from Prime Edited patient CD34 + cells had restored p47phox protein expression and NADPH oxidase activity to ~80% of normal healthy donors as determined by multiple ROS detection assays. To evaluate reproducibility, Prime Editors were delivered to CD34 + cells from 6 different healthy donors. On average, greater than 90% of CD34 + cells were Prime Edited across 6 donors in independent experiments. To confirm that Prime Edited CD34 + cells retain stem cell multipotency, engraftment, and hematopoietic reconstitution properties, Prime Edited and mock treated CD34 + cells from three different donors were transplanted into immunodeficient mice. Sixteen weeks after CD34 + cell transplantation, >87% of long-term hematopoietic stem cells (LT-HSC) that repopulated the bone marrow had at least 1 allele corrected. There were no significant differences between ‘electroporation only’ mock treated and Prime Editor electroporated human CD34 + cells with respect to long-term CD34 + cell engraftment, human CD45 + blood cell chimerism, hematopoietic multilineage blood cell reconstitution and biodistribution, or LT-HSC cell potency. Across these studies, >95% human CD45 + cell chimerism was achieved with Prime Edited CD34 + cells. Despite high on-target correction of the delGT mutation in NCF1, off-target editing, unintended editing, and chromosomal rearrangements (larger deletions or translocation) events were not detected using a robust suite of genome-wide assays. In summary, Prime Editing corrects a mutation that causes the most common autosomal recessive from of CGD; correction of this delGT mutation at NCF1 restores NADPH oxidase activity and function in myeloid progeny of Prime Edited CGD CD34 + cells; and Prime Edited long-term HSCs retain multipotency and functionality in vivo. These results support the development of a Prime Edited CGD patient autologous CD34 + cell therapy as a potential curative approach for p47phox-deficient CGD patients.

Post-Transplant Cyclophosphamide for the Prevention of Graft-vs.-Host Disease in Allogeneic Hematopoietic Cell Transplantation: A Guide to Management for the Advanced Practitioner.

Cyclophosphamide remains a critical component to haploidentical transplant conditioning regimens. Post-transplant cyclophosphamide (PTCy) emerged as an effective component of graft-vs.-host disease (GVHD) prophylaxis in the nonmyeloablative haploidentical bone marrow transplant setting. The relative ease of administration compared with ex vivo manipulations and efficacy in reducing GVHD has led to increasing PTCy use in transplant centers around the world. The role of PTCy has expanded to haploidentical transplantation with myeloablative conditioning regimens and peripheral blood progenitor cells as the donor source. Moreover, encouraging results in GVHD management have been shown with the use of PTCy alone or in combination with other immunosuppressives in the human leukocyte antigen-matched donor setting. The toxicity profile of cyclophosphamide varies extensively depending on dose, duration, overall drug exposure, and, potentially, pharmacogenetics. This review highlights the pharmacology, pharmacokinetics, and toxic effects of cyclophosphamide and offers practical guidance for clinical application in the post-transplant setting. We summarize data on the management of high-dose cyclophosphamide toxicities and provide insights into the pharmacogenetic implications on drug efficacy and safety data.

A case series of adrenoleukodystrophy in children conceived through in vitro fertilization with an egg donor

To report 3 cases of adrenoleukodystrophy (ALD) in children conceived by invitro fertilization (IVF) and egg donation. A case report. Patients aged 4-5 years old, evaluated by the University of Minnesota Leukodystrophy Center, who were diagnosed with ALD after being conceived by IVF with oocytes provided by the same donor. One patient received a hematopoietic stem cell transplant from a human leukocyte antigen-matched donor, and 1 patient received autologous lentiviral corrected hematopoietic cells. The disease state in 1 patient was unfortunately too advanced for effective treatment to be administered. Progression of disease after diagnosis or treatment was observed by cerebral magnetic resonance imaging and monitoring the development or advancement of any cognitive, adaptive, and motor deficits. Patients who received a transplant for ALD successfully experienced little to no disease progression at least 6 months to 1 year after treatment. These 3 cases of transmission of ALD through oocyte donation and IVF highlight the potential need to implement more comprehensive genetic screening of gamete donors to prevent the transfer of rare but severe genetic diseases through IVF. Further, these cases highlight limitations in carrier screening guidelines that limit reportable variants to pathogenic and likely pathogenic variants.

HLA gamma block matching in unrelated hematopoietic stem cell transplantation and the development of graft versus host disease

The human leukocyte antigen (HLA) genes routinely typed for hematopoietic stem cell transplantation (HSCT) are HLA-A, -B, -C, -DRB1 and -DQB1. HLA mismatches (MM), which have been associated with many post-transplantation complications, including acute graft-versus-host disease (GvHD), sometimes occur when a fully matched donor is not available. Gamma block (GB) is located in the central HLA region between Beta and Delta blocks and contains many inflammatory and immune regulatory genes, including the C4 gene. C4 was proposed as a marker when predicting haplotype matching due to positive linkage disequilibrium (LD) with its surrounding loci. Our aim was to investigate the association between GB matching in patients and their 9/10 HLA matched unrelated donors (UD) and the occurrence of GvHD. Patients and their UDs were typed using the PCR-SSP kit that detects 25 SNPs within GB. Gamma block mismatch occurred in 25 (75.8%) of the 33 studied patient-UD pairs. There was a significant difference in GvHD occurrence between Gamma type matched (GT-M) and mismatched (GT-MM) patient-UD pairs (p=0.0302). The probability of GvHD occurrence had also shown an increase, although insignificant, along with the number of GT-MM between patient-UD pairs (p=0.0913). These results suggest that GT matching could be useful in reducing the risk of post-HSCT complications.

Recruitment of Marrow Donors in the Non-governmental Sector of the Republic of North Macedonia, 2022

BACKGROUND: Bone marrow transplantation is a well-established lifesaving treatment procedure for many patients with various diseases, such as blood cell cancers (leukemia), some severe hematological diseases (aplastic anemia) and some rare diseases, as well as immune deficiency disorders. Allogeneic hematopoietic stem cell transplantation (HSCT) represents a potentially curative therapy for various hematologic disorders and even the only curative treatment for some diseases (e.g., myelodysplastic syndromes). As an alternative donor strategy, in patients lacking a human leukocyte antigen-matched donor or haploidentical donor, mismatched unrelated donors offer the option of using HSCT in this group of patients. Since the establishment of the first registry in the world for recruitment of unrelated bone marrow donors, the number of newly formed registries has increased over the years. In our country, in addition to the government-funded registry, housed at the Institute of Immunobiology and Human Genetics, Faculty of Medicine in Skopje, an additional non-governmental registry was established, Scientific Foundation SPIROSKI - Marrow Donor Registry (MK-SFSMDR). This will contribute to the increase of the number of newly registered donors in the Republic of North Macedonia and will be a great benefit for the patients searching for a bone marrow transplantation. AIM: We aimed to present the establishment of the first MDR in the non-governmental sector of the Republic of North Macedonia and the recruitment strategies of new donors. METHODS: The core activity was the creation, realization, and implementation of the Marrow Donors Recruitment Program. The constructed program for increasing the awareness of the population about HSCT and the importance of becoming a marrow donor encompasses the following activities: (1) creation of a web portal for bone MDR in English, Macedonian, and Albanian language; (2) application of standards from the World Marrow Donor Association for educational material preparation and informed consent creation; (3) composing printed and electronic materials for recruitment activities; (4) organization of public debates for marrow donation in governmental and non-governmental sectors; (5) inclusion of students and other non-governmental organizations for recruitment and registration of marrow donors; and (6) submission the obtained results from Macedonian donors and the donors from other communities (Albanians, Roma, Serbs, Turks, Vlachs, and others) in the database for marrow donors. RESULTS: In 2022, 988 submissions from marrow donors were registered, from which 678 (68.62%) were females and 310 (31.37%) were males. The biggest number of submissions for marrow donors are in the age group of 26–40 years (567 or 57.38%), followed by the age group of 18–25 (270 or 27.32%) and the age group of 41–55 (151 or 15.28%). The most efficient method for new submissions was through social networks, 538 or 54.45% out of 988 registered donors. The second was through word of mouth (200 or 20.24%), the third was onsite marrow donor recruitment actions (145 or 14.68%), followed by information from the websites (19 or 1.92%), and other (86 or 8.70%). From the total Macedonian population (2,062,044), we were able to recruit 988 marrow donors, which is 0.048% of the population. The biggest recruitment percentage was in the Skopje region (0.067%), followed by the Pelagonia region (0.059%) and the Southwest region (0.043%). The lowest participation was in the Polog region (0.011%). The biggest percentage of recruited marrow donors (988) were Macedonians (94.13%), and the rest of them were from the minorities: Albanians (1.92), Turks (0.71%), Roma (0.1%), Croats (0.2%), Bosniaks (0.51%), Vlachs (0.81%), Serbs (1.21%), Macedonian Muslims (0%), and others (0.4%). CONCLUSION: With the establishment of the SFS-MDR (MK-SFSMDR), one of the goals of SFS was achieved, which, together with the publication of scientific journals, enables and contributes to the scientific research in the field of the main tissue-compatible complex and transplantation of cells, tissues, and organs. The results will be used by patients who need bone marrow treatment in our country, in neighboring countries, and around the world.

Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott–Aldrich syndrome

Patients with Wiskott–Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

MiR-181a and -b expression in acute lymphoblastic leukemia and its correlation with acute graft-versus-host disease after hematopoietic stem cell transplantation, COVID-19 and torque teno viruses.

Acute lymphoblastic leukemia (ALL), a malignant transformation and proliferation of the lymphoid line of blood cells, is characterized by chromosomal abnormalities and genetic changes. The purpose of this research was the evaluation of expression level of miR-181a and -b in patients with ALL compared to the control group. Furthermore, we examined their expression level in hematopoietic stem-cell transplantation (HSCT) patients who developed acute graft-versus-host disease (aGVHD) in comparison with those without aGVHD and explore the relationship between their expression level and cytogenetic abnormalities. In this cross-sectional study, 76 newly diagnosed adult De novo ALL patients were enrolled who were admitted to our referral hospital. All patients received standard chemotherapy, consisting of daunorubicin. A total of 37 patients underwent HSCT from the related human leukocyte antigen-matched donors. ALL patients have been diagnosed with the coronavirus disease 2019 (COVID-19) and Torque teno viruses (TTVs). We assessed the expression levels of miR-181a and -b in the peripheral blood sample of ALL patients at the time of diagnosis prior to chemotherapy, and healthy matched individuals by RT–PCR. TTVs and COVID-19 load were also determined via RT–PCR. In conclusion, the expression level of miR-181a and -b were significantly higher in ALL patients than healthy controls and also increased in patients who developed aGVHD in comparison with those without aGVHD. MiR-181a and -b can be a useful biomarker in ALL and a useful indicator of aGVHD. The expression level of miR-181a in ALL patients with COVID-19 is significantly up-regulated, while it is reduced in these patients with TTV.

Haploidentical Hematopoietic Stem Cell Transplant Using Post-transplant Cyclophosphamide: A Single-Center Initial Experience in Vietnam.

Haploidentical transplants constitute a potential alternative therapy for patients who urgently need transplantation in the absence of human leukocyte antigen-matched donors. We report a single-center experience regarding the initial results of haploidentical peripheral blood stem cell transplantation (haplo-PBSCT) using post-transplant cyclophosphamide (PTCy) at the HCMC Blood Transfusion Hematology (BTH) Hospital. We conducted a retrospective case series study of 23 patients who underwent haplo-PBSCT using PTCy at the HCMC BTH Hospital between January 2014 and January 2021. The refined disease risk index (DRI) was used to stratify the outcomes. We evaluated the engraftment rate, graft-versus-host disease (GVHD), and complications during haploidentical transplantation. Overall survival (OS), disease-free survival (DFS), and GVHD-free relapse-free survival (GRFS) were assessed. The majority of the patients in the present study were diagnosed with acute myeloid leukemia. All patients received reduced-intensity conditioning regimens. The engraftment rate was 86.9%. The median times to neutrophil and platelet engraftment were 17 and 31 days, respectively. Two patients (8.7%) reported severe acute GVHD (grade III-IV), while two patients (8.7%) had grade I-II acute GVHD. Three patients experienced limited chronic GVHD of the skin, requiring topical steroids. The most common complication was bloodstream infection (60.9%). Cytomegalovirus reactivation occurred in 19 patients (82.6%) and 17.4% developed hemorrhagic cystitis. The 1-year relapse rate was 32.5%. The cumulative incidence of non-relapse mortality at 1 year was 17.3%. The 1-year OS and DFS rates were 66.3% and 55.7%, respectively. The 1-year GRFS rate was 49.2%. A high/very high DRI score was associated with worse OS after haplo-PBSCT (P=0.038). Haploidentical transplant using PTCy is a feasible therapy for patients without suitably matched donors in Vietnam. Infection after transplantation remains a challenge and requires effective management.

Combination of Low-Dose, Short-Course Mycophenolate Mofetil With Cyclosporine and Methotrexate for Graft-Versus-Host Disease Prophylaxis in Allogeneic Stem Cell Transplant.

With the standard regimen for graft-versushost disease prophylaxis in allogeneic stem cell transplant with human leukocyte antigen-matched donor, grade II-IV acute graft-versus-host disease occurs in 30% to 50% of sibling and up to 80% of unrelated recipients. Studies with limited patient numbers have shown efficacy and safety of mycophenolate mofetil for graft-versus-host disease prophylaxis. We investigated the effect of low-dose mycophenolate mofetil added to a standardized prophylaxis regimen for graft-versus-host disease in related human leukocyte antigen-matched allogeneic stem cell transplant. In this prospective randomized clinical trial, we compared cyclosporine and methotrexate versus the combination of cyclosporine, methotrexate, and mycophenolate mofetil in all patients who underwent human leukocyte antigencompatible related donor allogeneic stem cell transplant for acute leukemia during 3 years at the Bone Marrow Transplant Unit at Namazi Hospital, Shiraz University of Medical Sciences (Shiraz, Iran). All 134 patients in both groups underwent successful engraftment. Recovery times for neutrophils and platelets were not significantly different between groups (P < .05). Incidence of acute graft-versus-host disease in the cyclosporine, methotrexate, and mycophenolate mofetil group was less than in the cyclosporine and methotrexate group (21.6% vs 40.9%; P = .041). Incidence of grade II-IV acute graftversus-host disease in the mycophenolate mofetil group was 15.2% versus the control group at 33% (P = .045). Our single-center study suggests the combination of mycophenolate mofetil, cyclosporine, and methotrexate is superior to the standard regimen of cyclosporine and methotrexate for graft-versushost disease prophylaxis after human leukocyte antigen-matched related donor allogeneic stem cell transplant.

Adult and Cord Blood-Derived High-Affinity gB-CAR-T Cells Effectively React Against Human Cytomegalovirus Infections.

Human cytomegalovirus (HCMV) reactivations are associated with lower overall survival after transplantations. Adoptive transfer of HCMV-reactive expanded or selected T cells can be applied as a compassionate use, but requires that the human leukocyte antigen-matched donor provides memory cells against HCMV. To overcome this, we developed engineered T cells expressing chimeric antigen receptors (CARs) targeted against the HCMV glycoprotein B (gB) expressed upon viral reactivation. Single-chain variable fragments (scFvs) derived from a human high-affinity gB-specific neutralizing monoclonal antibody (SM5-1) were fused to CARs with 4-1BB (BBL) or CD28 (28S) costimulatory domains and subcloned into retroviral vectors. CD4+ and CD8+ T cells obtained from HCMV-seronegative adult blood or cord blood (CB) transduced with the vectors efficiently expressed the gB-CARs. The specificity and potency of gB-CAR-T cells were demonstrated and compared in vitro using the following: 293T cells expressing gB, and with mesenchymal stem cells infected with a HCMV TB40 strain expressing Gaussia luciferase (HCMV/GLuc). BBL-gB-CAR-T cells generated with adult or CB demonstrated significantly higher in vitro activation and cytotoxicity performance than 28-gB-CAR-T cells. Nod.Rag.Gamma (NRG) mice transplanted with human CB CD34+ cells with long-term human immune reconstitution were used to model HCMV/GLuc infection in vivo by optical imaging analyses. One week after administration, response to BBL-gB-CAR-T cell therapy was observed for 5/8 mice, defined by significant reduction of the bioluminescent signal in relation to untreated controls. Response to therapy was sporadically associated with CAR detection in spleen. Thus, exploring scFv derived from the high-affinity gB-antibody SM5-1 and the 4-1BB signaling domain for CAR design enabled an in vitro high on-target effect and cytotoxicity and encouraging results in vivo. Therefore, gB-CAR-T cells can be a future clinical option for treatment of HCMV reactivations, particularly when memory T cells from the donors are not available.

Unmanipulated Haploidentical Transplant Followed By Post-Transplant Cyclophosphamide and Selective Ex Vivo T-Cell Depleted Haploidentical Transplant Results in Comparable Outcome As Unrelated Cord Blood Transplant for Adults with Haematological Malignancies- a Multicenter Study in Singapore

Background: Outcomes after haploidentical (Haplo) haematopoietic cell transplantation (HCT) and after unrelated cord blood transplantation (UCBT) are encouraging and have become alternative options to treat patients with high-risk haematological malignancies without human leukocyte antigen (HLA) matched related or unrelated donor. There is paucity of data comparing the outcome of UCBT and haplo HCT. We retrospectively analyse and compare the outcome of adult patients with haematological malignancies receiving UCBT and haplo HCT using two different platform for graft-versus-host disease (GVHD) prophylaxis: selective ex-vivo T cell (TCRαβ and CD45RA+) depleted haplo HCT (Koh LP et al. Blood 2018; 132: 2093a) vs unmanipulated T cell replete haplo HCT with high dose posttransplant cyclophosphamide (PTCy). Methods: We studied 169 adults patients receiving allogeneic HCT using 4-6/6 HLA matched UCB (n=100) graft or Haplo (n=69) for various haematologic malignancies between Aug 2006 and July 2019, following myeloablative (MAC, n=76) or reduced intensity conditioning (RIC, N=93) regimen. 37 Haplo patients received unmanipulated non ex-vivo, T cell depleted graft followed by PT Cy for graft-versus-host disease (GVHD) prophylaxis (Haplo PTCy), whereas 32 patients received haplo-HCT with selective ex vivo T cell (TCRαβ and CD45RA+) depleted grafts for GVHD prophylaxis (Haplo-TCD). Results: Two year overall survival (OS) for patients undergoing UCB, Haplo PTCy and Haplo-TCD transplant were 46%, 54% and 55% (p=0.379), and event free survival (EFS) were 41%, 50% and 45% (p=0.573), respectively; these were not significantly different among the 3 groups. Two year cumulative incidence (C.I.) of non-relapse mortality (UCB 32% vs Haplo PT Cy 20% vs 31%; p=0.514), relapse-related mortality (RRM) (UCB 24% vs Haplo PT Cy 21% vs 16%; p=0.596) and grades 3 - 4 acute GVHD at 6 months (UCB 9% vs Haplo PT Cy 6% vs 10%; p=0.758) were not significantly different among the 3 groups. However, C.I. of chronic GVHD at 2 years was higher in PTCy as compared with others (Haplo PT Cy 28% vs UCB 4% and Haplo TCD 5%, respectively, P&lt;0.001). Multivariable analysis showed a significant association with OS and EFS for disease risk index (DRI) (p&lt;0.001) and HCT comorbidity index (p&lt;0.001), with no statistically significant impact from the type of stem cell graft used. In patients with low/intermediate risk DRI, the 2 year OS for TCD, PTCy and UCB were 71%, 55% and 56%, respectively (p=0.729), and the corresponding 2 year LFS were 59%, 54% and 48%, respectively (p=0.994). In patients with high/very high risk DRI, the 2 year OS for TCD, PTCy and UCB were 19 %, 51% and 18%, respectively (p=0.177), and the corresponding 2 year LFS were 0 %, 18% and 18%, respectively (p=0.774). Conclusions: Haploidentical HCT using either unmanipulated graft and PTCy or selective Ex Vivo TCRαβ and CD45RA+ depleted graft results in equivalent outcome to those HCT performed using UCB. It provides additional alternative for patients lacking HLA matched donors. Disclosures No relevant conflicts of interest to declare.

Availability of Multiple HLA-Matched Unrelated Donors for Allogeneic HCT Recipients

Introduction Success of allogeneic hematopoietic cell transplantation (HCT) is marred by adverse outcomes that include relapse of underlying malignancy, graft versus host disease (GVHD), and post-transplant viral infections. An ideal approach to minimize these adverse outcomes is to select an immunogentically ideal donor for each HCT recipient that not only has minimum allogeneic disparity with the recipient but also possesses an immunogenetic predisposition for strong anti-leukemic and anti-viral immune responses. In addition to human leukocyte antigen (HLA) compatibility, several other immunogenetic systems like killer Immunoglobulin-like receptors (KIRs) and variants in cytokine genes have shown promise in reducing HCT adverse outcomes. Their clinical utility in improving donor selection can only be assessed if for every patient there are more than one HLA matched donors available. Objectives In this study, a retrospective analysis of World Marrow Donor Association (WMDA) Search & Match Service search reports were performed for potential HCT recipients from a single center to assess how many allele level HLA matched donors were available for each recipient. Methods A retrospective, manual review of WMDA (formerly Bone Marrow Donors Worldwide) search reports for recipients seeking unrelated allogeneic HCT at Alberta Blood and Marrow Transplant Program in 2017 was performed (n=110). Patients were predominantly Caucasians. Allele-level match at HLA-A, HLA-B, HLA-C, and HLA-DRB1 was considered in determination of match level (n/8). The number of potential 8/8 and 7/8 matched unrelated donors were recorded. Donor prospects were categorized as ‘potential donor': total number of donors presented in WMDA search report; ‘high-probability donors': subset of donors known or highly-favored to match the patient; and ‘optimal donors': subset of high probability donors who were male and age ≤ 35. Results When 8/8 HLA matching was considered, more than one ‘potential', ‘high-probability' and ‘optimal' unrelated donors were available for 93%, 67% and 57% recipients respectively. When 7/8 HLA matching was considered, more than one ‘potential', ‘high-probability' and ‘optimal' unrelated donors were available for 100%, 98% and 94% recipients respectively. Conclusions Multiple HLA-matched unrelated donors are available for majority of HCT recipient of Caucasian ethnicity in BMDW search. These findings provide an ideal platform for prospective studies/clinical trials on unrelated HLA-matched HCTs to assess other non-HLA immunogenetic systems for finding an immunogenetically ideal donor (e.g., HLA matched and possessing favorable KIR or cytokine variants) for every HCT recipient.

Isolated Extramedullary Relapse in a Case of Acute Myeloid Leukemia Following Allogeneic Stem Cell Transplantation

Background: Acute myeloid leukemia (AML) is the commonest acute leukemia in adults. Allogeneic stem celltransplantation (ASCT) is a curative option for a subset of these patients.Case Presentation: We report the case of a 36 years old female patient who presented in April 2014 with pancytopenia.Investigations revealed that she had AML (M6). She achieved complete remission with induction chemotherapy which wasfollowed by four cycles of consolidation chemotherapy until a human leukocyte antigen-matched donor was available. Sheunderwent ASCT in January 2015. After 14 months, in March 2016, she presented with left breast and rightparapharyngeal masses. Histopathological examination of the excised mass showed infiltration with myeloid cells and thebone marrow was normocellular without leukemic infiltration. She received radiotherapy to the affected breast and thecervical region followed by chemotherapy with good response.Conclusion: AML relapse following ASCT may be in the form of isolated Extramedullary disease. Further research isneeded to optimize the management of these cases.

Have haploidentical transplants replaced umbilical cord transplants for acute leukemias?

Haploidentical stem cell transplantation (Haplo SCT) and umbilical cord blood stem cell transplantation (UCB SCT) have emerged over the past two to three decades as viable sources of alternative donor SCT when a human leukocyte antigen matched donor is not available. However, which of these two donor types is optimal for patients with leukemia in need of allografting is unknown. For patients with acute leukemia, results of UCB SCT have been improved by the use of double umbilical cord units and emerging ex-vivo expansion technologies. However, the costs associated with procuring double cord units and high transplant-related mortality due to delayed immunological reconstitution and infections, particularly in adult patients, remain a problem. Recently, Haplo SCT has become an increasingly utilized alternative donor source. While improvements of ex-vivo T-cell depletion platforms continue, emergence of T-cell-replete platforms, such as the use of post-transplantation cyclophosphamide (PTCy), is increasingly being utilized in treating acute leukemia patients. PTCy-based Haplo SCT is gaining popularity among transplant clinicians due to its relatively easy learning curve, low cost, low incidence of graft-versus-host disease, and favorable survival in acute leukemia patients. The clinical question of whether Haplo SCT should replace UCB SCT needs to be answered by ongoing randomized trials. However, the rapidly increasing adoption of Haplo SCT worldwide as the viable alternative for patients without a human leukocyte antigen-matched donor has seemingly addressed the question ahead of scientific judgment.

Third-party regulatory T cells prevent murine acute graft-versus-host disease.

Background/AimsAdoptive therapy with regulatory T (Treg) cells to prevent graft-versus-host disease (GVHD) would benefit from a strategy to improve homing to the sites of inflammation following hematopoietic stem cell transplantation (HSCT). Although donor-derived Treg cells have mainly been used in these models, third-party-derived Treg cells are a promising alternative for cell-based immunotherapy, as they can be screened for pathogens and cell activity, and banked for GVHD prevention. In this study, we explored major histocompatibility complex (MHC) disparities between Treg cells and conventional T cells in HSCT to evaluate the impact of these different cell populations on the prevention of acute GVHD, as well as survival after allogeneic transplantation.MethodsTo induce acute GVHD, lethally irradiated BALB/c (H-2d) mice were transplanted with 5 × 105 T cell-depleted bone marrow cells and 5 × 105 CD4+CD25– splenic T cells from C57BL/6 (H-2b) mice. Recipients were injected with 5 × 105 cultured donor-, host-, or third-party-derived CD4+CD25+CD62L+ Treg cells (bone marrow transplantation + day 1).ResultsSystemic infusion of three groups of Treg cell improved clinicopathological manifestations and survival in an acute GVHD model. Although donor-derived Treg cells were immunologically the most effective, the third-party-derived Treg cell therapy group displayed equal regulation of expansion of CD4+CD25+- Foxp3+ Treg cells and suppressive CD4+IL-17+ T-helper (Th17) cells in ex vivo assays compared with the donor- and host-derived groups.ConclusionsOur findings demonstrate that the use of third-party Treg cells is a viable alternative to donor-derived Treg cellular therapy in clinical settings, in which human leukocyte antigen-matched donors are not always readily available.

Generating autologous hematopoietic cells from human-induced pluripotent stem cells through ectopic expression of transcription factors.

Hematopoietic cell transplantation (HCT) is a successful treatment modality for patients with malignant and nonmalignant disorders, usually when no other treatment option is available. The cells supporting long-term reconstitution after HCT are the hematopoietic stem cells (HSCs), which can be limited in numbers. Moreover, finding an appropriate human leukocyte antigen-matched donor can be problematic. If HSCs can be stably produced in large numbers from autologous or allogeneic cell sources, it would benefit HCT. Induced pluripotent stem cells (iPSCs) established from patients' own somatic cells can be differentiated into hematopoietic cells in vitro. This review will highlight recent methods for regulating human (h) iPSC production of HSCs and more mature blood cells. Advancements in transcription factor-mediated regulation of the developmental stages of in-vivo hematopoietic lineage commitment have begun to provide an understanding of the molecular mechanism of hematopoiesis. Such studies involve not only directed differentiation in which transcription factors, specifically expressed in hematopoietic lineage-specific cells, are overexpressed in iPSCs, but also direct conversion in which transcription factors are introduced into patient-derived somatic cells which are dedifferentiated to hematopoietic cells. As iPSCs derived from patients suffering from genetically mutated diseases would express the same mutated genetic information, CRISPR-Cas9 gene editing has been utilized to differentiate genetically corrected iPSCs into normal hematopoietic cells. IPSCs provide a model for molecular understanding of disease, and also may function as a cell population for therapy. Efficient differentiation of patient-specific iPSCs into HSCs and progenitor cells is a potential means to overcome limitations of such cells for HCT, as well as for providing in-vitro drug screening templates as tissue-on-a-chip models.

Effect of HLA mismatching at HLA-A, -B, and -DRB1 for umbilical-cord blood transplantation in Taiwan

Unrelated cord blood transplantation has become a reliable alternative therapy for children and adults owing to that one or two antigen/allele mismatches between a patient and the cord blood donor are acceptable without occurrence of graft-versus-host disease. To investigate the relationship between the number and types of mismatches and relapse, we compared the number of mismatched and non-mismatched donor-recipient pairs, number of mismatched alleles, and number of mismatched antigens at each of the Human Leukocyte Antigen (HLA) -A, -B, and -DR loci, respectively. The result indicates that the number of mismatched antigens at the HLA-A locus was significantly associated with occurrence of relapse (X2P-value=0.0243; RR=1.49, 95% CI: 1.04–2.13). Additionally, the number of mismatched donor-recipient pairs and the number of mismatched alleles at the HLA-DR locus was negatively associated with risks of relapse (X2P-value=0.0028; RR=0.52, 95% CI: 0.31–0.89). In this study, we found that the mismatch at the HLA-A locus is associated with increased risk of relapse; while the mismatch at the HLA-DR locus is innocuous. Hence, we suggest that the well-matched HLA-A alleles were most critical for matching HLA alleles between umbilical-cord blood transplantation donors and recipients. In other words, cord blood transplantation requires less stringent HLA matching, if there are two 5/6 or 4/6 HLA matched donors, it's better to choose HLA-A matched donor at least.