Results of haploidentical transplant in patients with donor-specific antibodies: a survey on behalf of the Spanish Group of Hematopoietic Transplant and Cell Therapy.

Background: Donor-specific antibodies (DSAs) are IgG allo-antibodies against mismatched donor HLA molecules and can cause graft failure (GF) in the setting of haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Our aim was to report the experience of the Spanish Group of Hematopoietic Transplant (GETH-TC) in DSA-positive patients who had undergone haplo-HSCT. Methods: We conducted a survey of patients who underwent haplo-HSCT in GETH-TC centers between 2012 and 2021. Data were collected on the DSA assay used, monitoring strategy, complement fixation, criteria for desensitization, desensitization strategies and transplant outcomes. Results: Fifteen centers from the GETH-TC responded to the survey. During the study period, 1,454 patients underwent haplo-HSCT. Seventy of the transplants were performed in 69 DSA-positive patients, all of whom lacked a suitable alternative donor; 61 (88%) patients were female (90% with prior pregnancies). All patients received post-transplant cyclophosphamide-based graft-versus-host disease prophylaxis. Regarding baseline DSA intensity, 46 (67%) patients presented mean fluorescence intensity (MFI) >5,000, including 21 (30%) with MFI >10,000 and three (4%) with MFI >20,000. Six patients did not receive desensitization treatment, four of them with MFI <5,000. Of 63 patients receiving desensitization treatment, 48 (76%) were tested after desensitization therapy, and a reduction in intensity was confirmed in 45 (71%). Three patients (5%) experienced an increase in MFI after desensitization, two of whom experienced primary GF. Cumulative incidence of neutrophil engraftment at day 28 was 74% in a median of 18 days (IQR, 15─20); six patients died before engraftment due to toxicity or infection and eight patients had primary GF despite desensitization in seven of them. After a median follow-up of 30 months, two-year overall and event-free survival were 46.5% and 39%, respectively. The two-year cumulative incidence of relapse was 16% and non-relapse mortality (NRM) was 43%. Infection was the most frequent cause of NRM, followed by endothelial toxicity. Multivariate analysis identified baseline MFI >20,000 as an independent risk factor for survival and an increase in titers after infusion as an independent risk factor for GF. Conclusions: Haplo-HSCT is feasible in DSA-positive patients, with high rates of engraftment after desensitization guided by DSA intensity. Baseline MFI >20,000 and increased intensity after infusion are risk factors for survival and GF.

Familial CD45RA- T cells to treat severe refractory infections in immunocompromised patients.

Background: Immunocompromised patients are susceptible to high-risk opportunistic infections and malignant diseases. Most antiviral and antifungal drugs are quite toxic, relatively ineffective, and induce resistance in the long term. The transfer of pathogen-specific Cytotoxic T-Lymphocytes has shown a minimal toxicity profile and effectiveness in treating Cytomegalovirus, Adenovirus, Epstein - Barr virus, BK Virus and Aspergillus infections, but this therapy have the main limitations of regulatory issues, high cost, and absence of public cell banks. However, CD45RA- cells containing pathogen-specific memory T-cells involve a less complex manufacturing and regulatory process and are cheaper, feasible, safe, and potentially effective. Methods: We present preliminary data from six immunocompromised patients: four who had severe infectious diseases and two who had EBV lymphoproliferative disease. All of them underwent multiple safe familial CD45RA- T-cell infusions as adoptive passive cell therapy, containing Cytomegalovirus, Epstein - Barr virus, BK virus, and Aspergillus-specific memory T-cells. We also present the method for selecting the best donors for CD45RA- cells in each case and the procedure to isolate and store these cells. Results: The infusions were safe, there was no case of graft-versus host disease, and they showed a clear clinical benefit. The patients treated for BK virus nephritis, Cytomegalovirus encephalitis, Cytomegalovirus reactivation, and disseminated invasive aspergillosis experienced pathogen clearance, complete resolution of symptoms in 4-6 weeks and a lymphocyte increase in 3 of 4 cases after 3-4 months. Donor T cell transient microchimerism was detected in one patient. The two patients treated for EBV lymphoproliferative disease underwent chemotherapy and several infusions of CD45RA- memory T-cells containing EBV cytotoxic lymphocytes. Donor T-cell microchimerism was observed in both patients. The viremia cleared in one of the patients, and in the other, despite the viremia not clearing, hepatic lymphoproliferative disease remained stable and was ultimately cured with EBV-specific Cytotoxic T-Lymphocytes. Conclusion: The use of familial CD45RA- T-cells containing specific Cytotoxic T-lymphocytes is a feasible, safe and potential effective approach for treating severe pathogen infections in immunocompromised patients through a third party donor. Furthermore, this approach might be of universal use with fewer institutional and regulatory barriers.

Identification of the novel HLA-C*08:243 allele in a Spanish bone marrow donor.

HLA-C*08:243 differs from C*08:51 by a single nucleotide substitution in codon 156 (G > T).

Haploidentical hematopoietic stem cell transplantation in pediatric and adolescent patients: A study of the Spanish hematopoietic stem cell transplantation group (GETH).

INTRODUCTION: The main advantages of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) are the immediate availability of donors, the possibility of developing cell therapy approaches with different novel transplant platforms, and the procedure's cost savings. METHODOLOGY: We retrospectively analyzed the pediatric haplo-HSCT activity of the Spanish hematopoietic stem-cell transplantation group (GETH) between 1999 and 2016, aiming to study clinical characteristics and outcomes by describing patient groups with non-malignant disease (NMD) or malignant disease (MD) and the impact of 2 different periods (1999-2009 and 2010-2016) on long-term outcomes. RESULTS: Twelve centers performed 232 haplo-HSCTs in 227 children, representing 10% of all pediatric allogeneic HSCT activity in Spain from 1999 to 2016, with a notable increase since 2013. Most haplo-HSCTs (86.7%) were performed in patients with MD; 95% received peripheral blood stem cells from donors, and 78.9% received ex vivo T-cell depleted grafts. Non-manipulated grafts using post-transplantation cyclophosphamide have been incorporated since 2012. We observed a higher percentage of graft failure in NMD versus MD (32% vs. 15.6%; p=0.029). Relapse and transplant-related mortality were the procedure's main limitations in MD and NMD, respectively. Five-year overall survival was 48.5% (SE 3.9), with no statistically significant difference when comparing the MD and NMD cohorts. Patients who received previously a HSCT the overall survival was significantly decreased. We observed no survival improvement over time. CONCLUSIONS: Although haplo-HSCT is an increasingly employed treatment option, our patients' results need improvement. We need to develop reference centers, especially for NMD whose rarity makes it difficult to gain experience.

Management of Donor-Specific Antibodies in Haploidentical Transplant: Multicenter Experience From the Madrid Group of Hematopoietic Transplant.

Background: Donor specific antibodies (DSAs) can be responsible for graft failure (GF) in the setting of mismatched hematopoietic stem cell transplantation (HSCT). The aim of our study is to report the experience of the Madrid Group of Hematopoietic Transplant (GMTH) in patients with DSAs undergoing haplo-HSCT. Methods: Patients undergoing haplo-HSCT in centers from the GMTH from 2012 to 2020 were included in the study. DSAs were analyzed with a solid-phase single-antigen immunoassay; monitoring was performed during desensitization on days -14, -7, 0 and in a weekly basis until neutrophil engraftment. Desensitization strategies varied depending on center experience, immunofluorescence intensity, complement fixation and type of antibodies. Results: We identified a total of 20 haplo-HSCT in 19 patients performed with DSAs in 5 centers. 10 (53%) patients presented anti-HLA class I DSAs (6 of them with > 5000 mean fluorescence intensity (MFI)), 4 (21%) presented anti-HLA class II (1 with > 5000 MFI) and 5 (26%) presented both anti-HLA class I and II (5 with > 5000 MFI). 90% of patients received at least two treatments as desensitization strategy and all experienced a decrease of MFI after desensitization (mean reduction 74%). Only one patient who developed progressive increase of MFI after infusion developed GF. Desensitization treatments used included rituximab, immunoglobulins, therapeutic plasma exchange, incompatible platelets, buffy coat and immunosuppressors. Seventeen (90%) patients achieved neutrophil engraftment; one patient died before engraftment because of infection and one patient with class I DSAs developed primary GF despite an intensive desensitization. After a median follow-up of 10 months, OS and EFS were 60% and 58%, respectively, cumulative incidence of relapse was 5% and NRM was 32%. Conclusions: Despite the optimal strategy of DSAs desensitization remains unclear, the use of desensitization treatment guided by DSAs intensity kinetics constitute an effective approach with high rates of engraftment for patients with DSAs in need for an haplo-HSCT lacking an alternative suitable donor.

Evaluation of the Spanish population coverage of a prospective HLA haplobank of induced pluripotent stem cells.

BACKGROUND: iPSC (induced pluripotent stem cells) banks of iPSC lines with homozygous HLA (human leukocyte antigen) haplotypes (haplobanks) are proposed as an affordable and off-the-shelf approach to allogeneic transplantation of iPSC derived cell therapies. Cord blood banks offer an extensive source of HLA-typed cells suitable for reprogramming to iPSC. Several initiatives worldwide have been undertaken to create national and international iPSC haplobanks that match a significant part of a population. METHODS: To create an iPSC haplobank that serves the Spanish population (IPS-PANIA), we have searched the Spanish Bone Marrow Donor Registry (REDMO) to identify the most frequently estimated haplotypes. From the top ten donors identified, we estimated the population coverage using the criteria of zero mismatches in HLA-A, HLA-B, and HLA-DRB1 with different stringencies: high resolution, low resolution, and beneficial mismatch. RESULTS: We have calculated that ten cord blood units from homozygous donors stored at the Spanish cord blood banks can provide HLA-A, HLA-B, and HLA-DRB1 matching for 28.23% of the population. CONCLUSION: We confirm the feasibility of using banked cord blood units to create an iPSC haplobank that will cover a significant percentage of the Spanish and international population for future advanced therapy replacement strategies.

The role of early natural killer cell adoptive infusion before engraftment in protecting against human herpesvirus-6B encephalitis after naïve T-cell-depleted allogeneic stem cell transplantation.

BACKGROUND: Naïve T-cell-depleted grafts have been employed as an ex vivo T-cell depletion (TCD) platform to prevent graft-versus-host disease (GvHD) and improve immune reconstitution by providing rapid donor memory T-cell reconstitution after allogenic hematopoietic stem cell transplantation (allo-HSCT). CD45RA- memory T cells confer protection against viruses such as cytomegalovirus, Epstein-Barr virus, and adenovirus; however, reports have shown an unexpectedly high incidence of human herpesvirus (HHV)-6B encephalitis among pediatric allo-HSCT patients. METHODS: We report the first 18 consecutive allo-HSCT, 16 haplo-HSCT, and two human leukocyte antigen-matched related donors implanted with naïve TCD grafts. All donors were administered three cell products: first, a CD34+ stem cell product; second, a CD45RA+ TCD graft, followed by an adoptive natural killer (NK) cell infusion within 10 days after HSCT. The study's primary endpoint was the incidence of HHV-6B encephalitis. RESULTS: Engraftment was achieved in 94.5% of cases; 2-year overall survival, event-free survival, and GvHD/relapse-free survival were 87.2% (95% CI 78.6-95.8), 67.3% (95% CI 53.1-81.5), and 64% (95% CI 50.5-78.1), respectively. HHV-6B reactivation occurred in 7 of the haplo-HSCT patients, six of who received a cell infusion with an NK/CD4 ratio <2. None of the patients developed encephalitis. CONCLUSIONS: In this clinical study, we show that early adoptive NK cell infusion after a 45RA+ TCD allo-HSCT graft is safe and can prevent HHV-6B encephalitis. We recommend infusing adoptive NK cells after allo-HSCT using CD45RA+ TCD grafts.

Phase 2 Clinical Trial of Infusing Haploidentical K562-mb15-41BBL-Activated and Expanded Natural Killer Cells as Consolidation Therapy for Pediatric Acute Myeloblastic Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) accounts for approximately 20% of pediatric leukemia cases; 30% of these patients experience relapse. The antileukemia properties of natural killer (NK) cells and their safety profile have been reported in AML therapy. We proposed a phase 2, open, prospective, multicenter, nonrandomized clinical trial for the adoptive infusion of haploidentical K562-mb15-41BBL-activated and expanded NK (NKAE) cells as a consolidation strategy for children with favorable and intermediate risk AML in first complete remission after chemotherapy (NCT02763475). PATIENTS AND METHODS: Before the NKAE cell infusion, patients underwent a lymphodepleting regimen. After the NKAE cell infusion, patients were administered low doses (1 × 106/IU/m2) of subcutaneous interleukin-2. The primary study endpoint was AML relapse-free survival. We needed to include 35 patients to demonstrate a 50% reduction in relapses. RESULTS: Seven patients (median age, 7.4 years; range, 0.78-15.98 years) were administered 13 infusions of NKAE cells, with a median of 36.44 × 106 cells/kg (range, 6.92 × 106 to 193.2 × 106 cells/kg). We observed chimerism in 4 patients (median chimerism, 0.065%; range, 0.05-0.27%). After a median follow-up of 33 months, the disease of 6 patients (85.7%) remained in complete remission. The 3-year overall survival was 83.3% (95% confidence interval, 68.1-98.5), and the cumulative 3-year relapse rate was 28.6% (95% confidence interval, 11.5-45.7). The study was terminated early because of low patient recruitment. CONCLUSION: This study emphasizes the difficulties in recruiting patients for cell therapy trials, though NKAE cell infusion is safe and feasible. However, we cannot draw any conclusions regarding efficacy because of the small number of included patients and insufficient biological markers.

Haploidentical transplantation in pediatric non-malignant diseases: A retrospective analysis on behalf of the Spanish Group for Hematopoietic Transplantation (GETH).

OBJECTIVE: Describe the GETH haploidentical stem cell transplantation (haplo-HSCT) activity in non-malignant disease (NMDs). METHODS: We retrospectively analyzed data from children with NMDs who underwent haplo-HSCT. RESULTS: From January 2001 to December 2016, 26 pediatric patients underwent 31 haplo-HSCT through ex vivo T cell-depleted (TCD) graft platforms or post-transplantation cyclophosphamide (PT-Cy) at 7 Spanish centers. Five cases employed unmanipulated PT-Cy haplo-HSCT, 16 employed highly purified CD34+ cells, and 10 employed ex vivo TCD grafts manipulated either with CD3+ CD19+ depletion, TCRαß+ CD19+ selection or naive CD45RA+ T-cell depletion. Peripheral blood stem cells were the sole source for patients following TCD haplo-HSCT, and bone marrow was the source for one PT-Cy haplo-HSCT. The most common indications for transplantation were primary immunodeficiency disorders (PIDs), severe aplastic anemia, osteopetrosis, and thalassemia. The 1-year cumulative incidence of graft failure was 27.4%. The 1-year III-IV acute graft-versus-host disease (GvHD) and 1-year chronic GvHD rates were 34.6% and 16.7%, respectively. The 2-year overall survival was 44.9% for PIDs, and the 2-year graft-versus-host disease-free and relapse-free survival rate was 37.6% for the other NMDs. The transplantation-related mortality at day 100 was 30.8%. CONCLUSION: Although these results are discouraging, improvements will come if procedures are centralized in centers of expertise.

Description of a novel HLA null allele, DRB1*15:176N.

A new HLA null allele, DRB1*15:176N, was characterized in a Spanish volunteer bone marrow donor.

Somatic mutation in the two HLA-B genes of a patient with acute myelogenous leukemia.

In this report, we describe a case of somatic mutations in the two HLA-B genes in a patient with acute myelogenous leukemia. The HLA-B*15:01 allele showed an insertion of two nucleotides within exon 2 leading to a premature stop codon. HLA-B*40:01 showed one nucleotide substitution within exon 3, identical to that described for B*15:258N. The restriction of these mutations in leukemic cells was confirmed in patient's samples from buccal epithelial cells and hematopoietic cells obtained when the patient was in remission. The clinical significance of somatic HLA mutations in cancer seems to be associated with escape from immune surveillance and clonal evolution. The analysis of possible mutations in HLA genes of tumor cells would be valuable information for the outcome of the disease and stem cell donor selection.

HLA-B*56:55:01:02, -C*03:374 and -DPB1*13:01:03 characterized by next-generation sequencing.

The new HLA alleles HLA-B*56:55:01:02, -C*03:374 and -DPB1*13:01:03 were characterized by NGS methodology.

Analysis of the Spanish CCR5-∆32 inventory of cord blood units: lower cell counts in homozygous donors.

The possibility to use CCR5-∆32 umbilical cord blood to cure HIV infection in patients in need of a hematopoietic transplant has been suggested. The less stringent HLA compatibility needed in this type of transplant facilitates the search of a suitable donor having the CCR5-∆32 mutation. To achieve an inventory of CCR5-∆32 cord blood units, the 20,236 best cell quality units of the Spanish Registry were genotyped. Furthermore, their CD34+ and total nucleated cells counts, blood type, gender, HLA and donor's geographical and ancestral origin were analyzed. The results showed 130 (0.64%) units homozygous for the deletion, 2,646 (13.08%) heterozygous and 17,460 (86.28%) did not present the mutation. Interestingly, a significant lower amount of CD34+ cells was found in the CCR5-∆32 homozygous units. In addition, a significant association was found among donor's ancestral origin and the mutation, with a higher percentage of CCR5-∆32 units with a European ancestry. In summary, identification of a relatively high number of CCR5-∆32 units is feasible and will facilitate the development of clinical trials for HIV cure in patients requiring hematopoietic transplantation. Further studies are required to understand the significance of lower cell counts within the CCR5-∆32 homozygous group and its clinical impact.

Mesenchymal stem cells are of recipient origin in pediatric transplantations using umbilical cord blood, peripheral blood, or bone marrow.

Chimerism status of bone marrow (BM)-derived mesenchymal stem cells (MSC) from children who received an allogeneic hematopoietic transplant is an interesting question, referring not only to hematopoietic transplant but also to cellular therapy. MSC are present in the BM but it is still controversial whether these cells maintain their capacity for homing to the BM. In this report, we studied chimerism of BM-derived MSC from children who underwent a hematopoietic stem cell transplant using BM, peripheral blood, and umbilical cord blood as source of hematopoietic stem cells.

Transient donor cell-derived myelodysplastic syndrome with monosomy 7 after unrelated cord blood transplantation.

Donor cell leukaemia or myelodysplastic syndromes are extremely rare complications that have been observed not only after haematopoietic transplantation with progenitor cells harvested from bone marrow and peripheral blood, but also after cord blood transplantation. We describe the early onset of monosomy 7 in donor cells after cord blood transplantation in a patient diagnosed with myelodysplastic syndrome 3 months after transplantation. Fluorescent in situ hybridisation analysis performed in a cryopreserved aliquot of the cord blood showed 2.5% of nuclei with monosomy 7. The cord blood donor was studied and he showed neither peripheral blood cytopenias nor cytological or cytogenetic features of myelodysplasia. The cell blood counts (CBC) of the girl have improved over 2 yr while decreasing the percentage of monosomic cells. The monosomic clone has finally disappeared and the CBC are finally normal. This case of transient monosomy 7 started very early after engraftment emphasises the relevance of clonal instability of specific progenitor cells in the early engraftment, and host immune status, in cytogenetic abnormalities founded in donor cell-derived MDS and acute leukaemia. Moreover, the clinical follow-up of this patient, recommends a more conservative treatment for this clonal disease early developed after transplantation.

Bi-directional allelic recognition of the human minor histocompatibility antigen HB-1 by cytotoxic T lymphocytes.

Human minor histocompatibility antigens (mHag) are target antigens of the graft-versus-leukemia response observed after allogeneic HLA-identical stem cell transplantation. We previously defined the molecular nature of the B cell lineage-specific mHag HB-1. The CTL epitope was identified as the decamer peptide EEKRGSLHVW presented in the context of HLA-B44. The HB-1 antigen is encoded by a locus of yet unknown function on chromosome 5q32. A single nucleotide polymorphism within this locus results in an amino acid change from histidine (H) to tyrosine (Y) at position P8 within the CTL epitope. Based on genomic information, we have developed a PCR-RFLP assay to perform HB-1 typing at the DNA level. We determined that the allelic frequency for the H and Y variant is 0.79 and 0.21, respectively. From these data, we calculated that the expected recipient disparity between HLA-B44-matched sibling pairs for HB-1H is 2.8%, whereas recipient disparity for HB-1Y is expected to be 12.4%. Therefore, we addressed whether the HB-1Y peptide is reciprocally immunogenic. We revealed that both peptide variants bind equally efficient to HLA-B44 molecules and that the H/Y substitution has no influence on formation of epitope precursor peptides by 20 S proteasome-mediated degradation. More directly, CTL recognizing the naturally presented HB-1Y peptide could be generated from a HB-1H homozygous donor using peptide-pulsed dendritic cells. Using a set of synthetic structurally related peptide variants, we found that the H/Y substitution has a major impact on TCR recognition by CTL specific for either of the HB-1 allelic homologues. HB-1 is the first human mHag described that induces bi-directional allogeneic CTL responses that may contribute to a specific graft-versus-leukemia response following allogeneic stem cell transplantation.

Efficient and nontoxic adenoviral purging method for autologous transplantation in breast cancer patients.

Tumor cell contamination of clinical grafts is a major concern in autologous hematopoietic stem cell transplantation because these contaminating cells can contribute to relapse. In the present work, we use a suicide gene therapy approach that successfully accomplishes the two main goals of any purging strategy: highly efficient elimination of contaminating tumor cells and preservation of the engraftment capability of the hematopoietic progenitor cells. Human CD34(+) cells spiked with breast cancer cells were infected with an adenoviral vector encoding the cytosine deaminase transgene (Ad-CMV-CD). In vitro, transduction with Ad-CMV-CD followed by exposure to 400 micro M 5-fluorocytosine resulted in complete elimination of clonogenic contaminating tumor cells without affecting the clonogenic potential of the human hematopoietic CD34(+) cells. Transplantation of nonobese diabetic/LtSz-scid/severe combined immunodeficient (NOD/SCID) mice with nonpurged contaminated grafts and purged contaminated grafts, allowed us to test the safety and efficacy of our procedure in two independent purging experiments. Hematopoietic engraftment kinetics as well as the quantity and quality of human engraftment were not affected by the purging therapy. Results showed a significant difference in survival between the nonpurged group (28%) and the purged group (100%; P = 0.012). Moreover, highly sensitive histological and molecular analyses confirmed the absence of tumor cells in the recipients of purged marrow. In contrast, metastatic tumors were detected in animals that received nonpurged grafts. We anticipate that this strategy will result in a safe and efficacious hematopoietic graft product for autologous transplantation for patients with multiple forms of epithelial cancers.