TYPE Original Research PUBLISHED 26 May 2023 DOI 10.3389/fimmu.2023.1165759 Results of haploidentical OPEN ACCESS transplant in patients with EDITED BY Eric Spierings, Utrecht University, Netherlands donor-specific antibodies: a REVIEWED BY Shigeo Fuji, survey on behalf of the Spanish Osaka International Cancer Institute, Japan Luca Castagna, Azienda Ospedaliera Ospedali Riuniti Villa Group of Hematopoietic Sofia Cervello, Italy *CORRESPONDENCE Transplant and Cell Therapy Mi Kwon mi.kwon@salud.madrid.org Rebeca Bailén Rebeca Bailén 1,2*, Raquel Alenda 3, Beatriz Herruzo-Delgado 4, rebeca.bailen@salud.madrid.org Cynthia Acosta-Fleitas 5, Ana Vallés 6, Albert Esquirol 7, RECEIVED 14 February 2023 Marta Fonseca 8, Laura Solán 9, Irene Sánchez-Vadillo 10, ACCEPTED 16 May 2023 PUBLISHED 26 May 2023 Guiomar Bautista 11, Leyre Bento 12, Oriana López-Godino 13, CITATION Ariadna Pérez-Martı́nez 14, Anna Torrent 15, Joud Zanabili 16, Bailén R, Alenda R, Herruzo-Delgado B, Marı́a Calbacho 17, Miguel Ángel Moreno 3, Acosta-Fleitas C, Vallés A, Esquirol A, Fonseca M, Solán L, Sánchez-Vadillo I, Marı́a Jesús Pascual-Cascón 4, Luisa Guerra-Domı́nguez 5, Bautista G, Bento L, López-Godino O, Pérez-Martı´nez A, Torrent A, Zanabili J, Anabelle Chinea 6, Irene Garcı́a-Cadenas 7, Lucı́a López-Corral 8, Calbacho M, Moreno MÁ, Pascual- Francisco Boix-Giner 8,18, José Luis López Lorenzo 9, Cascón MJ, Guerra-Domı´nguez L, Chinea A, Garcı´a-Cadenas I, López- Karem Humala 10, Rafael Duarte 11, Antonia Sampol 12, Corral L, Boix-Giner F, López Lorenzo JL, Humala K, Duarte R, Sampol A, Heras I, Inmaculada Heras 13, José Luis Vicario 3, Antonio Balas 3, Vicario JL, Balas A, Oarbeascoa G, Gillen Oarbeascoa 1,2, Paula Fernández-Caldas 1, Fernández-Caldas P, Anguita J and Kwon M (2023) Results of haploidentical Javier Anguita 1,2,19 and Mi Kwon 1,2,19* on behalf of Grupo transplant in patients with donor-specific antibodies: a survey on behalf of the Español de Trasplante Hematopoyético y Terapia Celular Spanish Group of Hematopoietic (GETH-TC) Transplant and Cell Therapy. Front. Immunol. 14:1165759. 1 Department of Hematology and Hemotherapy, Hospital General Universitario Gregorio doi: 10.3389/fimmu.2023.1165759 Marañón, Madrid, Spain, 2 Translational Oncology Section, Gregorio Marañón Health Research COPYRIGHT Institute, Madrid, Spain, 3 Department of Histocompatibility, Centro de Transfusión de la Comunidad © 2023 Bailén, Alenda, Herruzo-Delgado, de Madrid, Madrid, Spain, 4 Department of Hematology and Hemotherapy, Hospital Universitario Acosta-Fleitas, Vallés, Esquirol, Fonseca, Regional de Málaga, Málaga, Spain, 5 Department of Hematology and Hemotherapy, Hospital Solán, Sánchez-Vadillo, Bautista, Bento, Universitario Doctor Negrı´n, Gran Canaria, Spain, 6 Department of Hematology and Hemotherapy, López-Godino, Pérez-Martı´nez, Torrent, Hospital Universitario Ramón y Cajal, Madrid, Spain, 7 Hematology Department, Hospital de la Santa Zanabili, Calbacho, Moreno, Pascual-Cascón, Creu i Sant Pau, Sant Pau Health Research Institute and Jose Carreras Leukemia Research Institutes, Guerra-Domı´nguez, Chinea, Garcı´a- Universitat Autonoma of Barcelona, Barcelona, Spain, 8 Department of Hematology and Hemotherapy, Cadenas, López-Corral, Boix-Giner, López Hospital Clı´nico Universitario de Salamanca, Salamanca, Spain, 9 Department of Hematology and Lorenzo, Humala, Duarte, Sampol, Heras, Hemotherapy, Hospital Universitario Fundación Jiménez Dı´az, Madrid, Spain, 10Department of Vicario, Balas, Oarbeascoa, Fernández- Hematology and Hemotherapy, Hospital Universitario La Paz, Madrid, Spain, 11 Department of Caldas, Anguita and Kwon. This is an open- Hematology and Hemotherapy, Hospital Universitario Puerta de Hierro Majadahonda, Madrid, Spain, access article distributed under the terms of 12 Department of Hematology and Hemotherapy, Hospital Universitario Son Espases, Palma de the Creative Commons Attribution License Mallorca, Spain, 13 Department of Hematology and Hemotherapy, Hospital Universitario Morales (CC BY). The use, distribution or Meseguer, Murcia, Spain, 14 Department of Hematology and Hemotherapy, Hospital Clı´nico reproduction in other forums is permitted, Universitario de Valencia, Valencia, Spain, 15 Department of Hematology and Hemotherapy, Hospital provided the original author(s) and the Germans Trias i Pujol, Barcelona, Spain, 16 Department of Hematology and Hemotherapy, Hospital copyright owner(s) are credited and that Universitario Central de Asturias, Asturias, Spain, 17 Department of Hematology and Hemotherapy, the original publication in this journal is Hospital Universitario 12 de Octubre, Madrid, Spain, 18 CIBERONC and Centro de Investigación del cited, in accordance with accepted Cáncer-Instituto de Biología Molecular y Celular del Cáncer (Universidad de Salamanca - CSIC), academic practice. No use, distribution or Salamanca, Spain, 19 Department of Medicine, Universidad Complutense de Madrid, Madrid, Spain reproduction is permitted which does not comply with these terms. Frontiers in Immunology 01 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 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. KEYWORDS donor-specific antibodies, graft failure, haplo-HSCT, desensitization strategies, DSA kinetics Introduction HSCT) is growing every year (2). However, these transplant modalities have introduced new challenges, including the Allogeneic hematopoietic stem cell transplantation (allo-HSCT) strategies required in the presence of donor-directed anti-human is the only curative treatment option for several hematological leukocyte antigen (HLA)-specific allo-antibodies (DSAs). disorders, mainly malignant diseases (1). For those patients who DSAs are preformed mostly IgG antibodies with specificity lack an HLA-identical sibling or matched unrelated donor (MUD), against HLA molecules not shared with the donor (3). Their the use of alternative donor strategies, including mismatched significance in mediating rejection was first described in solid unrelated donors (MMUDs), umbilical cord blood (UCB) and organ transplantation, and has been widely reported to be related haploidentical donors, has significantly increased the possibility of to graft failure (GF) in several HSCT settings, including MUD, allo-HSCT. In fact, the number of haploidentical HSCT (haplo- MMUD, UCB and haplo-HSCT (3–9). Accordingly, screening for Frontiers in Immunology 02 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 DSAs before these procedures is essential (10). DSAs are more committee of the Hospital General Universitario Gregorio Marañó n. frequently reported in the haplo-HSCT setting, due to a higher All patients signed informed consent. degree of mismatch and, in the case of female recipients, to the possibility of alloimmunization to offspring antigens following pregnancy (11). HLA typing and DSA identification The gold standard technique for the detection of DSAs is based on solid-phase immunoassays (SPI) using the Luminex® platform (12). HLA typing was performed at high/intermediate resolution using Although non-quantitative, this approach measures the DNA-based techniques (20) in two different samples. Haploidentical immunofluorescence intensity of the antibodies, expressed as mean donors were all related donors sharing a single HLA haplotype; patients fluorescence intensity (MFI), which has been found to have a clinically were studied 10 loci (HLA-A, -B, -C, -DRB1 and -DQB1). Patient sera significant association with graft failure (GF) (13). It can also be was collected from clotted samples to perform Luminex® SPI IgG modified to detect complement-fixing DSAs (C1q+ or C3d) (14). In single antigen tests (Lifecodes, Immucor, USA or One Lambda, DSA-positive patients, the first option should be to search for donors ThermoFisher Scientific Brand, USA), covering HLA-A, -B, -C, against whom the recipient does not show DSAs. However, this option -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1 and -DPB1 is not always available, and in those cases where alternative suitable antigens (12). Units were expressed as raw MFI and normalized MFI donors cannot be found, desensitization strategies should be considered respectively. Some patients were also tested for complement-fixing if therapeutic options other than transplantation are not possible. In antibodies using C1q or C3d techniques. For data analysis, IgG MFI recent years, different groups have reported multimodal desensitization values between 1,000 and 5,000 were considered low; between 5,000 strategies based on those developed for solid organ transplantation (10, and 10,000 intermediate; and >10,000 high, as reported by the EBMT 11, 14–16), including a combination of strategies that remove consensus (11). (therapeutic plasma exchange [TPE]) or neutralize (incompatible platelet or buffy coat transfusion) preformed antibodies, reduce their production (rituximab [RTX], bortezomib or immunosuppressive DSA monitoring and desensitization therapies), or inhibit the complement cascade (IV immunoglobulins strategy [IVIG]). The use of these techniques has reduced the incidence of GF to less than 10% (17). However, no study has compared the different The survey included policies followed for both DSA monitoring combination strategies, and the procedure depends on center policies and the desensitization strategy used. DSA screening was performed and experience. Only one set of consensus guidelines has been once the mismatched HSCT was indicated. Some centers also analyzed published thus far by the European Society for Blood and Marrow additional samples prior to desensitization, prior to infusion and/or Transplantation (EBMT), in 2018 (11). More recently, the MD post infusion according to their policies and experience. Anderson Cancer Center (MDACC) and City of Hope group has Desensitization strategies used also varied according to center reported the outcomes of an homogeneously-desensitized cohort of 37 policies and experience, immunofluorescence intensity, type of DSA-positive patients who underwent haplo-HSCT previously treated antibodies (anti-HLA class I and/or anti-HLA class II) and with TPE, IVIG, RTX and buffy coat (18), comparing them with those complement fixation, if available. Treatments included RTX, IVIG, of a haplo-HSCT cohort without DSAs. TPE, incompatible platelet transfusions, buffy coat transfusion, Recently, the Madrid Group of Hematopoietic Transplant immunosuppressive agents (mycophenolate mofetil [MMF], (GMTH) reported the effect of desensitization treatments guided tacrolimus and steroids) and bortezomib. The combination of by DSA intensity kinetics in a cohort of 19 patients (19). In this treatment used and decision on whether to treat or not depended on regard, the aim of the present study within the Spanish Group of the center policy and was analyzed separately. Hematopoietic Transplant and Cell Therapy (GETH-TC) was to confirm these results in a large cohort of patients across Spain. Pre- and post-transplant evaluation Patients and methods Patients were stratified according to the disease risk index (21). Pre-transplant comorbidities were evaluated using the HSCT Patients and variables comorbidity index (HCT-CI) (22). Acute graft-versus-host disease (aGVHD) was scored according to MAGIC criteria (23), and A survey was sent to all centers belonging to the GETH-TC to chronic GVHD (cGVHD) was scored according to the NIH identify patients with DSAs who underwent haploidentical donor Consensus Development Project (24). Myeloid engraftment was transplantation. Information was collected on DSA screening, defined as an absolute neutrophil count (ANC) of 0.5×109/L or monitoring strategy and desensitization strategy used, together with greater for three consecutive days. Platelet engraftment was defined pre-transplant characteristics and outcomes. The study period included as a platelet count of 20×109/L or higher without transfusion support patients transplanted between January 2012 and December 2021. for three consecutive days. Patients who survived more than 28 days Disease and transplant characteristics were collected from the and failed to achieve myeloid engraftment were evaluated on a case-by- electronic records of each center. The study was conducted case basis to discard possible graft failure. Diagnosis of disease according to the Declaration of Helsinki and approved by the ethics recurrence was based on clinical and pathological criteria. Frontiers in Immunology 03 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 Study variables The characteristics of the 69 patients and transplants are shown in Table 1. Sixty-one patients (88%) were female, and 90% of them The primary endpoints were rates of myeloid and platelet had prior pregnancies. All patients had received multiple engraftment. Secondary endpoints included occurrence of transfusions prior to transplant. The most frequent diagnoses aGVHD, cGVHD, endothelial toxicity, relapse, non-relapse were acute myeloid leukemia and high-risk myelodysplastic mortality (NRM) or death from any cause. Relapse, toxic death syndrome. Patients with prior transplant relapsed after the first and second transplant due to GF were considered events for event- transplant and had an urgent indication for a second procedure. All free survival (EFS). Analysis performed for overall survival (OS), patients lacked an alternative donor against whom no DSAs were EFS, relapse, NRM, GF and endothelial toxicity included the HSCT present; haplo donors were selected according to MFI and donor period (2013─2017 vs. 2018─2021), patient and donor sex, patient characteristics, prioritizing younger donors. Most patients age, number of pregnancies (0, 1─2, >2), prior HSCT, HCT-CI received PBSCs. (0─2 vs. >2), cytomegalovirus (CMV) sero-status (iso vs. no, donor Myeloablative conditioning regimens were used in 32 (46%) negative/recipient positive vs. other), ABO incompatibility (none patients, including FluBux regimen in 19 (fludarabine [Flu] 40 mg/ vs. minor vs. major), intensity of conditioning regimen m2/day on days -6 to -3 plus 3 or 4 days of IV busulfan [Bux] 3.2 (myeloablative vs. reduced-intensity), stem cell source (peripheral mg/kg/day on days -6 to -3); ten received the TBF regimen (Flu, blood stem cells [PBSC] vs. bone marrow [BM]), CMV reactivation Bux, thiotepa [TT] 5 mg/kg/day (2-3 days)) and three received total before day 100, DSA class (I vs. II vs. both), baseline intensity of body irradiation [TBI]-based regimens with either Flu, VP-16 or DSAs (<5,000 vs. 5,000─9,999 vs. 10,000─19,999 vs. >20,000 MFI), cyclophosphamide (Cy). Thirty-five patients (51%) received a baseline complement fixation, increase after infusion and use of a reduced intensity conditioning (RIC) regimen: 17 with a modified desensitization strategy (none, RTX-based, non RTX-based). Last Baltimore protocol consisting of Flu 30 mg/m2/day days -6 to -2, Cy update of the cohort was performed in December 2021. 14.5 mg/kg/day on days -6 and -5 and Bux 3.2 mg/kg/day either one or two days on days -3 and -2 (one patient received TBI instead of Bux); 17 patients received a RIC TBF regimen and one patient was Statistical analysis conditioned with clofarabine and melphalan (CloMel). Two patients received a sequential transplant due to active disease with Qualitative variables were expressed as frequency and percentage. Clo and Ara-C followed by RIC conditioning. Quantitative variables were expressed as median and either All patients received GVHD prophylaxis with high-dose post- interquartile range (IQR) (25th and 75th percentiles) or range. The transplant Cy 50 mg/kg/day on days +3 and +4 together with MMF c2 test was used to identify correlations between qualitative variables, 10 mg/kg/day (except for five patients) and either tacrolimus (46 and the non-parametric Kruskal-Wallis test was used for quantitative patients, 67%) or cyclosporine A (23 patients, 33%) since day +5. variables. Variables that were significantly correlated in the univariate Three patients (4%) also received ATG. analysis were evaluated using a forward stepwise selection method with a p-in value of <0.05 and a p-out of <0.1. The criterion for inclusion in multivariate Cox regression analysis was a p-value of <0.1. Estimates of DSA characteristics, kinetics EFS and OS were calculated using the Kaplan-Meier method. and management Cumulative incidence curves and competing risk regression were performed as alternatives to Cox regression for survival data in the Characteristics and kinetics of DSAs are shown in Table 2. At presence of competing risks. In our case, the only competing event for baseline, 46 patients (67%) presented intensity >5,000 MFI engraftment was toxic death prior to day 28. NRM and relapse were including 21 (30%) with intensity >10,000, of whom three (4%) considered competing events for each other, in addition to a second presented intensity >20,000 MFI. Complement fixation techniques transplant for both of them. Univariate and multivariate analyses were were performed in 20 patients (29%) and showed fixation in 14. A performed with SPSS (IBM, SPSS Statistics for Windows, Version 21.0. total of 63 patients (91%) received some desensitization treatment. Armonk, NY, USA) and Stata 17 for Windows. Cumulative incidence Reasons for not performing desensitization treatment included low was calculated with R Studio version 1.0.2. intensity of DSAs (4 patients with intensity <5,000 and 1 with a limit intensity of 6,800) and lack of specific guidelines in one patient who was transplanted in 2014. In the remaining 63 patients, Results desensitization treatment was used, including 19 patients with MFI <5,000. Patient and transplant characteristics Combinations of desensitization treatment used are described in Supplementary Figure 1. Treatments used included weekly RTX 375 Between January 2012 and December 2021, 1,454 patients mg/m2 in 53 patients (84% of those treated) (median 2 doses, range underwent haplo-HSCT in 15 centers in Spain. Among those, a 1─6); IVIG 0.4 mg/kg/day in 42 (67%) (median 5 days, range 1─20); total of 69 patients with DSAs underwent haplo-HSCT from a TPE in 33 (52%) (median 3 sessions, range 1─10); incompatible donor against whom DSAs were present in the recipient, with a total platelet transfusion only if class I DSAs were present on days -1 and/ of 70 HSCTs performed between November 2013 and July 2021. or 0 in 26 (41%, median 4.5 pools, range 1─10); MMF 5-10 mg/kg/bid Frontiers in Immunology 04 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 TABLE 1 Patient and transplant characteristics. TABLE 2 Donor specific antibodies: characteristics, kinetics and management. Patients (n=69) Patients (n=69) Sex (female, %) 61 (88) Baseline DSA characteristics (n, %) Age (median, IQR) 55 (46-61) • DSA anti-MHC class I only 33 (48) • Intensity >5,000 MFI 24 Diagnosis (n, %): • DSA anti-MHC class II only 19 (27) • AML/MDS 49 (71.5) • Intensity >5,000 MFI 7 • ALL 8 (12) • DSA anti-MHC class I and II 17 (25) • Non-Hodgkin lymphoma 5 (7) • Intensity >5,000 MFI 15 • Hodgkin lymphoma 4 (6) • Plasma cell leukemia 1 (1.5) Baseline DSA intensity (n, %) • Myelofibrosis 1 (1.5) • >5,000 MFI 46 (67) • Severe aplastic anemia 1 (1.5) • >10,000 MFI 21 (30) • >20,000 MFI 3 (4) Disease risk index (n, %) • Low 4 (6) Complement fixation techniques 20 (29) • Intermediate 35 (50.5) available (n, %) • High/Very High 29 (42) • Positive C1q/C3d fixation 14 • Not-applicable 1 (1.5) Patients receiving desensitization (n, HCT-CI score (n, %) %) 63 (91) • 0-2 36 (52) • Rituximab 53 (84) • ≥3 33 (48) • IVIG 42 (67) • TPE 33 (52) Sensitization events (n, %) • Incompatible platelet transfusion 26 (41) • Polytransfusion only 14 (20) • MMF 26 (41) • 1-2 pregnancies + polytransfusion 39 (57) • Tacrolimus 13 (21) • 3-4 pregnancies + polytransfusion 16 (23) • Buffy coat 12 (19) Prior HSCT (n, %) • Bortezomib 2 (3) • Autologous 4 (6) • Steroids 1 (2) • Allogeneic 4 (6) Monitoring performed after (% of 63 desensitized patients) • MSD 1 desensitization (n, %) • MUD 2 • Patients tested after desensitization 48 (76) • Haploidentical 1 • Patients with reduction in 45 (71) Donor (n, %) intensity • Sibling 27 (40) • Median reduction in intensity 100 (12─100) • Child 37 (53) (median %, range)* • Parent 3 (4) • Persistent DSA >5,000 MFI at 11 (17) • 2nd degree relative 2 (3) infusion • Persistent DSA >10,000 MFI at 3 (5) Stem cell source PB (n, %) 62 (90) infusion • Increase after infusion 3 (5) Graft counts • CD34+ cells (x106/kg) (median, 6.6 (2.5─15) DSA, donor-specific antibodies; IVIG, intravenous immunoglobulin; MFI, mean fluorescence range) (PB) intensity; MHC, major histocompatibility complex; MMF, mycophenolate mofetil; TPE, therapeutic plasma exchange. • TNC (x108/kg) (median, range) 2.7 (1.5─6.7) (BM) in 26 (41%) (median 14 days, range 7─35, starting 2 to 4 weeks before Conditioning regimen intensity (n, %) • Myeloablative 32 (46) the infusion date and until day -2); buffy coat only if class II DSAs were • Reduced intensity 35 (51) present on day -1 in 12 (19%); tacrolimus 0.06 mg/kg/day in 13 (21%); • Sequential regimen 2 (3) bortezomib in 2 (3%); and dexamethasone in 1 (2%). CMV serostatus (n, %) Most treatment combinations were personalized, and strategies • Donor and recipient positive 49 (71) varied between centers and depending on the period and DSA • Donor and recipient negative 4 (6) • Donor negative, recipient positive 15 (22) characteristics. Regarding patients with intensity <5,000 MFI (23 • Donor positive, recipient negative 1 (1) patients, 33%), four (17%) did not receive desensitization, five (22%) received only one treatment strategy without combination ABO incompatibility (n, %) • None 53 (77) and 14 (61%) received 2-3 treatments combined. Among those with • Major 6 (9) intensity >5,000 MFI (46, 67%), 44 (95%) received desensitization, • Minor 9 (13) with at least two treatments in 40 (87%). • Bidirectional 1 (1) After or during desensitization therapy, DSAs were monitored ALL, acute lymphoid leukemia; AML, acute myeloid leukemia; BM, bone marrow; CMV, cytomegalovirus; HSCT, hematopoietic stem cell transplant; HCT-CI, Hematopoietic Cell in 48 patients (76% of those desensitized) prior to infusion on days Transplantation-Comorbidity Index; IQR, interquartile range; MDS, myelodysplastic -1 or 0. Forty-five (71%) showed a reduction in intensity, with a syndrome; MSD, matched sibling donor; MUD, matched unrelated donor; PB, peripheral median reduction in intensity of 100% (range 12─100%, mean blood; TNC, total nucleated cell count. One patient with DSAs underwent two transplants; the second procedure is not described. 73%). Eleven (17%) patients showed persistent intensity of >5,000 Frontiers in Immunology 05 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 MFI at infusion and three (5%) of >10,000. Post-infusion Survival, relapse and non-relapse mortality monitoring was performed in 14 patients (22% of those desensitized). Three patients (5%) showed an increase in After a median follow-up of 30 months (range 4.5─97 months), immunofluorescence after infusion since day 0, two of whom the two-year overall survival (OS) and event-free survival (EFS) developed primary GF requiring a second salvage HSCT. were 46.5% (and 39%respectively (Figure 3). In the multivariate analysis, DSA intensity >20,000 MFI was identified as an independent risk factor for OS (adjusted pseudo-hazard ratio Engraftment and graft failure (asHR) 1.72 (95% CI, 1.1─2.9), p = 0.046); use of a RIC regimen was the only independent risk factor identified for EFS (asHR 2.11 Cumulative incidence of neutrophil recovery at day 28 was 74% (95% CI, 1.2─3.8), p = 0.013). Two-year survival rates were 65%, and at day 60 was 80% (Figure 1). Fifty-five (79%) patients achieved 39%, 42% and 0% for patients with baseline DSA intensity of <5,000 myeloid engraftment in a median of 18 days (range 12─46; IQR MFI, 5,000─10,000 MFI, 10,000─20,000 MFI and >20,000 15─20 days). Four of these patients engrafted later than day 28; MFI, respectively. three of them had shown severe complications that explained the Cumulative incidence of relapse (CIR) at 2 years was 16% and delay in engraftment, including infection and endothelial toxicity. cumulative incidence of NRM at 2 years was 42.6%. The Six patients (9%) died without engraftment prior to day 28 due to multivariate analysis identified both use of a RIC regimen (asHR toxicity. The cumulative incidence of platelet engraftment at day 28 2.26 (95% CI, 1.1─4.8), p = 0.032) and female donor (asHR 2.23 was 35% and 65% at day 100. Forty-eight (69%) patients achieved (95% CI, 1─5), p = 0.048) as risk factors for relapse, while no platelet engraftment in a median of 31 days (range 11-292, IQR 21- independent risk factors were identified for NRM. 54). Two patients developed secondary GF in the context of CMV A total of 41 (59%) patients died during the study period infection and 16 (23%) met criteria of poor graft function (Supplementary Table 1). Seven of the eight patients who after engraftment. experienced primary GF died (12%): four underwent a second Eight patients (12%) developed primary GF, six of them despite haplo-HSCT, one of them with DSAs against the donor; two died desensitization treatment. Regarding potential causes of primary GF due to complications of the second procedure; and one engrafted and in those patients who presented this complication (Figure 2), we was alive at last follow up. Ten patients (17%) died due to relapse of the identified absence of desensitization therapy in two out of eight underlying disease. Nine patients (22%) died as a result of infection not (25%), of whom one showed high intensity DSAs and was not related to GVHD (7 bacterial, 2 viral). Nine patients (22%) died desensitized and the other showed limit intensity of 6,800; neither of because of endothelial and CNS complications, including four these patients was tested for complement fixation. Among the other patients with sinusoidal obstruction syndrome (SOS), two with six patients, two (25%) showed MFI <5,000 (complement was not hemorrhage (one CNS hemorrhage in the context of platelet tested), one with DSAs against class I and the other against class II. transfusion refractoriness and one because of diffuse alveolar One of them received a bone marrow graft with a total nucleated hemorrhage), one with transplant-associated microangiopathy, one count under 2x108/kg; none of them engrafted at day 28 although with capillary leak syndrome and one because of CNS demyelinating both were rescued, one after infusion of a CD34+ selected boost and disease. Finally, six patients (15%) died due to GVHD, three because of the other with a second haplo donor against whom no DSAs were refractoriness to treatment and three due to infection under intensive present at that time (alive at last follow-up). Regarding the four immunosuppressive treatment. patients (50%) who had MFI >5,000 and primary GF despite desensitization, complement was tested in one (which showed fixation), three presented anti-class I DSAs, and one both anti- Toxicity and GVHD class I and II. Despite intensive desensitization with at least three treatments including RTX in all of them, one patient showed an Bacterial infections occurred before day 30 in 23 patients (33%); increase in intensity after the initial decrease and values <5,000 at 18 of them were bloodstream infections. Two patients (3%) were infusion, while one patient showed a prozone phenomenon, with diagnosed with breakthrough invasive fungal infection and 24 saturation of the technique at infusion that prevented detection of (35%) presented early viral reactivations including CMV and BK very high intensity DSAs at infusion. All four patients died, one virus-related cystitis. Forty-five patients (65%) experienced at least prior to salvage therapy because of bleeding and three after a second one CMV reactivation in the first 6 months after HSCT. salvage HSCT because of infection and respiratory failure, one of Endothelial complications were diagnosed in 14 patients (20%), them after initial engraftment. including ten patients (15%) who met criteria for SOS (one mild In the multivariate analysis performed for GF, increased case related to prior inotuzumab ozogamicin treatment and nine intensity after infusion was identified as an independent risk moderate to very severe cases accounting for four related deaths) factor (adjusted odds ratio (aOR) 24.6 (95% CI, 1.6-365), p = and four cases (6%) of TA-TMA (one patient died due to TMA and 0.020) (Table 3). two patients died with a concomitant diagnosis of SOS and CMV Frontiers in Immunology 06 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 A B C FIGURE 1 Engraftment. (A) Neutrophil engraftment. (B) Platelet engraftment. (C) Neutrophil engraftment by DSA baseline intensity. disease). Finally, one patient (1.5%) presented severe capillary leak Discussion syndrome, developing toxic epidermal necrolysis and died. We did not identify any independent risk factor for the development of DSAs play a major role as a risk factor for GF in mismatched endothelial toxicity in the multivariate analysis performed. HSCT, which presents as one of the barriers for haplo-HSCT (5, 14, Four patients (6%) developed neurological complications, 25). Until the publication of the EBMT guidelines in 2018 (11), including two patients who developed posterior cord syndrome there was a lack of recommendations for DSA management and during CMV reactivations, one patient who presented monitoring that have led to heterogeneous management in this demyelinating disease of the CNS and one patient who setting. Thus, centers have based their policies on their own developed Guillain-Barré -like demyelinating polyneuropathy of experience, the desensitization approaches described by groups unknown origin, achieving a complete response after with more experience, and the accessibility of treatments and IVIG treatment. monitoring techniques. Verifying the results of these policies is The cumulative incidence of grade II-IV aGVHD at day 180 both pertinent and useful to improve the strategies in this poorly was 29% and grade III-IV aGVHD was 13% at day 180. described population, and to identify risk factors for GF and Cumulative incidence of chronic GVHD at 10 months was survival. In an attempt to report the current practice and 25%, with 15% cumulative incidence of moderate-severe outcomes of haplo-HSCT in DSA-positive recipients in Madrid cGVHD (Supplementary Figure 2). (Spain), our group has recently published the experience of the Frontiers in Immunology 07 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 A Paents with DSA n=69 No Yes Desensizaon strategy used n=63 n=6 Reason Outcome Low Lack of 6/63 toxic 51/63 engraed 6/63 primary GF Intensity guidelines deaths prior to • 34 MFI >5000 n=5 n=1 (Pts 1, 2, 3, 5, 6, 7) day 28 and • 17 MFI <5000 engrament 1/5 primary 1/1 primary GF (Pt 4) GF (Pt 8) B FIGURE 2 Primary graft failure. Flow of patients according to baseline MFI and desensitization therapy. MFI, mean fluorescence intensity; DSA, Donor specific antibodies; GF, Graft failure; Pt, patient; MDS-EB, myelodysplastic syndrome with excess blasts; CR, complete remission; AML, acute myeloid leukemia; T-MDS, therapy-related MDS; TNC, total nucleated count; MAC, myeloablative conditioning; RIC, reduced intensity conditioning; RTX, rituximab; IVIG, intravenous immunoglobulins; TPE, therapeutic plasma exchange; IS, immunosuppressors; HSCT, hematopoietic stem cell transplantation; CNS, central nervous system; LFU, last follow-up. GMTH in a cohort of 19 patients, with similar results in terms of relatively low number of patients monitored and it should be engraftment and survival to those reported in unmanipulated confirmed in other studies, this fact highlights the importance of haplo-HSCT (19). Following this analysis, we aimed to expand MFI monitoring after infusion and the need for a continuous the cohort by including all Spanish patients reported to the GETH- desensitization strategy for these patients, as previously reported TC registry. To the best of our knowledge, this cohort gathers the (10); this should be considered as most centers do not have access largest reported experience of haplo-HSCT in patients with DSAs. to DSA intensity results in a timely manner. Moreover, the previously Although monitoring and desensitization strategies were mostly reported cut-off level for baseline intensity of >20,000 as a risk factor personalized, with both intra- and inter-center heterogeneity, some for survival (18) was also confirmed in our cohort. Therefore, in consistent data were found in our study. First, the incidence of patients with this very high intensity DSA, alternatives including engraftment in our cohort was similar to that reported by other change of donor should be examined. The possibility of using highly groups in patients with DSAs receiving a homogeneous MMUD against whom no antibodies are present should be desensitization strategy (18). Our patients were mostly treated considered in this very high-risk population (26, 27). Finally, based on baseline MFI, with a trend towards a more intensive regarding complement fixation, in those patients who were studied, approach in patients with MFI >5,000. Although other previously nearly 75% showed complement fixation. Moreover, in the three described important information including data on complement patients with GF and intensity <5,000 MFI, additional factors such as fixation and post-desensitization intensity was missing in a number not-detected complement fixation could have been the underlying of cases, the use of a desensitization approach for patients with cause of non-engraftment. Thus, if this technique is not available for intensity >5,000 might have mitigated the detrimental effect of patients with MFI <5,000, an assumption of positive fixation might be baseline DSA intensity in our cohort, highlighting the importance a safer approach than avoiding desensitization. Our rate of graft of this cut-off level. Second, a post-infusion increase in titers was failure in this population with <5,000 MFI is higher than that identified as an independent risk factor for GF in our cohort. reported by other authors that have reported an absence of impact Although this result should be taken with caution due to the of these low intensity levels of engraftment (28). However, we should Frontiers in Immunology 08 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 TABLE 3 Results of univariate and multivariate analyses. Overall survival Graft failure Variables sHR p- asHR p- p- aOR p- OR (95%CI) (95%CI) value (95%CI) value value (95%CI) value 1.09 Period (2018─2021) 0.801 – - 1.46 (0.26─8.07) 0.665 – - (0.54─2.24) 0.82 Sex (male) 0.709 – – 0.97 (0.10─9.37) 0.986 – - (0.29─2.31) 0.83 Prior HSCT 0.749 – – - - – - (0.27─2.59) 1.10 HCT-CI ≥3 0.758 – – 1.5 (0.34─6.71) 0.596 – – (0.60─2.05) 0.67 Donor sex (male) 0.193 – – 1.20 (0.26─5.59) 0.818 – – (0.36─1.23) 0.75 Iso-CMV sero-status 0.461 – - 0.75 (0.12─4.29) 0.747 – – (0.35─1.61) 1.12 Source (bone marrow) 0.820 – - 4.25 (0.63─28.7) 0.138 – – (0.43─2.94) 1.87 1.83 Reduced intensity conditioning 0.044 0.052 3.6 (0.66─19.7) 0.140 – – (1.02─3.44) (0.99─3.68) Rituximab-based desensitization 1.03 0.956 0.63 (0.21─1.84) 0.396 treatment (0.28─3.75) 0.97 Pregnancies (>2) 0.940 - - 1.00 (0.15─6.42) 1.000 – – (0.42─2.22) 0.73 Major ABO incompatibility 0.649 - - 1 (0.32─12.1) 0.452 – – (0.20─2.73) 0.92 CMV reactivation 0.839 - - 0.78 (0.07─8.43) 0.842 – – (0.45─1.92) 0.89 0.78 (0.47─1.69) 0.732 0.791 DSA class (I vs. II vs. both) - - (0.12─44.84) – – 0.71 0.464 0.904 0.89 (0.14─5.58) (0.29─1.74) 2.92 1.72 DSA intensity >20,000 MFI at baseline 0.001 0.046 3.33 (0.22─50.1) 0.384 – – (1.51─5.63) (1.09─2.94) 2.53 Complement-fixation at baseline 0.300 - - 0.57 (0.06─5.30) 0.622 – – (0.44─14.6) 1.13 DSA intensity >5,000 MFI at infusion 0.765 - - 2.62 (0.49─13.9) 0.257 – – (0.50─2.57) 1.32 24.67 24.67 Intensity increase after infusion 0.599 - - 0.020 0.020 (0.46─3.82) (1.67─365) (1.67─365) Values are expressed as absolute numbers and percentages, and hazard/odds ratio and 95% confidence interval. Significant differences are shown in bold. Cox regression analysis was adjusted by the most significant clinical characteristics using a forward stepwise method (see Statistical Analysis section). Criteria for inclusion of covariates in the multivariate analysis were p-value < 0.1. 95%CI, 95% confidence interval; sHR, pseudo hazard ratio; asHR, adjusted pseudo hazard ratio; OR, odds ratio; aOR, adjusted odds ratio; p-value, level of significance. Significant values are marked in bold. also address there was one case of primary GF in a patient with MFI < 20,000 MFI and use of a RIC regimen; the second factor might be 5,000 who received a suboptimal bone marrow graft with a total biased due to the high proportion of patients with prior HSCT, nucleated count under 2x108/kg, highlighting the importance of a older age and poorer HCT-CI in that group, which may have sufficient cell dose, especially in this setting. affected the analysis of OS, EFS and NRM. However, only three Regarding transplant outcomes, although the OS of our cohort patients presented baseline intensity > 20,000 MFI and this result was similar to that reported by other groups (18, 19), we found a should also be validated in further studies. The impact of DSAs, relatively high NRM rate as compared with the classic rates reported their intensity and the desensitization strategy on NRM and in haplo-HSCT with post-transplant Cy (29), mainly because of endothelial toxicity has been studied, but no relationship has been infection and endothelial toxicity or bleeding, excluding patients found. Interestingly, the proportion of patients with endothelial with GF. Factors related to OS were baseline intensity of DSAs > toxicity in our cohort appeared to be higher than that reported in Frontiers in Immunology 09 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 A B C D E FIGURE 3 Survival, relapse and non-relapse mortality. (A) Overall survival. (B) Event-free survival. (C) Non-relapse mortality. (D) Relapse. (E) Overall survival by DSA baseline intensity. haplo-HSCT (30, 31); however, the heterogeneity of the population could be hypothesized, we could not attribute any effect. Further and other concomitant risk factors such as prior drugs used may studies are needed to study the impact of desensitization on GVHD affect this result. Multivariate analysis was performed to identify development. Finally, we also found delayed platelet engraftment, specific risk factors related to DSAs, but we were unable to detect with only 35% patients engrafting at day 28 and 64% at day 100; this any impact. Despite this high rate of NRM could be circumstantial, delayed engraftment in patients with DSAs was also reported in the we cannot discard a possible effect of the presence of DSAs or their Madrid cohort and the MDACC experience (18, 19). Whether treatment. Thus, it should be considered in patients with a DSAs have played a role in this delay can be hypothesized but possibility of finding a donor against whom no DSAs are present, not proven in the setting of haplo-HSCT with post-transplant Cy, in especially in those with MFI > 20,000. On the other hand, the which poor graft function has also been reported (25). Neutrophil incidence of GVHD was lower than expected with a low related engraftment was also delayed in four patients, but may be at least mortality. While possible impact of the desensitization strategy partially explained by concomitant severe complications. Frontiers in Immunology 10 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 Among the limitations of this study, the design of the survey might Author contributions have introduced some bias, including selection bias, and it also has all the intrinsic limitations of retrospective studies. Both the population Conception and design: RB and MK. Provision of study and protocols included were heterogeneous, and the long study period materials or patients: All authors. Data collection: RB, RA, BH-D, also introduces heterogeneity, as there was a complete lack of guidelines CA-F, AV, AE, MF, LS, IS-V, GB, LB, OL-G, AP-M, AT, JZ and at the beginning of the period. Nevertheless, our study includes a large MC. Data analysis and interpretation: All authors. Manuscript cohort of patients with a very detailed report of monitoring performed writing: RB and MK. Revision and final approval of manuscript: and desensitization strategies used, analysis of possible GF causes, and All authors. All authors contributed to the article and approved the data on toxic complications, infections and GVHD. All this submitted version. information validates the risk factors that have previously been described and the usefulness of the recently published guidelines. Our report indicates a need to unify strategies, as previously reported Acknowledgments by part of our group in the Madrid experience (19). Analysis of this experience could also guide the creation of local guidelines for the Special thanks to the staff and nurses of all the hematology and management of these patients. Moreover, the validation of the transplant units for their care and contributions to this study. We importance of very high intensity DSAs and their impact on survival thank José Marı́a Belló n for the data analysis. We would also like to underlines the need for alternative strategies in patients with baseline thank Laura Hidalgo, Ph.D. (Medical Science Consulting, Valencia, intensity over 20,000 MFI. Spain) for her assistance with the preparation of the manuscript In conclusion, the optimal strategy for DSA desensitization is (financed by Jazz Pharmaceuticals as part of the “Publibecas” grant still unclear. Accordingly, the risk factors described in recent program of the GETH-TC). guidelines and publications including the importance of DSA intensity, persistence or increase in intensity at infusion and complement fixation should be taken into account in the Conflict of interest personalized strategy applied to each patient to make this approach safe for patients who lack an alternative donor. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Data availability statement The original contributions presented in the study are included Publisher’s note in the article/Supplementary Material. Further inquiries can be All claims expressed in this article are solely those of the authors directed to the corresponding author. and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its Ethics statement manufacturer, is not guaranteed or endorsed by the publisher. The studies involving human participants were reviewed and approved by COMITÉ de É TICA DE LA INVESTIGACIÓ N con Supplementary material MEDICAMENTOS HOSPITAL GENERAL UNIVERSITARIO GREGORIO MARAÑÓ N. Written informed consent for The Supplementary Material for this article can be found online at: participation was not required for this study in accordance with https://www.frontiersin.org/articles/10.3389/fimmu.2023.1165759/ the national legislation and the institutional requirements. full#supplementary-material References 1. Snowden JA, Sá nchez-Ortega I, Corbacioglu S, Basak GW, Chabannon C, de la 4. Ottinger HD, Rebmann V, Pfeiffer KA, Beelen DW, Kremens B, Runde V, et al. Camara R, et al. 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Recommendations for donor human leukocyte antigen assessment bloodadvances.2019000143 Frontiers in Immunology 12 frontiersin.org Bailén et al. 10.3389/fimmu.2023.1165759 Glossary Allo-HSCT Allogeneic hematopoietic stem cell transplantation BM Bone marrow CMV Cytomegalovirus DSA Donor-directed anti-HLA-specific allo-antibody EFS Event-free survival GETH-TC Spanish Group of Hematopoietic Transplant and Cell Therapy GF Graft failure GMTH Madrid Group of Hematopoietic Transplant GVHD Graft-versus-host disease Haplo-HSCT Haploidentical hematopoietic stem cell transplantation HCT-CI Hematopoietic stem cell transplant comorbidity index IVIG Intravenous immunoglobulins HLA Human leukocyte antigen MFI Mean fluorescence intensity MUD HLA-matched unrelated donor MMUD HLA-mismatched unrelated donor NRM Non-relapse mortality OS Overall survival PBSC Peripheral blood stem cells SOS sinusoidal obstruction syndrome TPE Therapeutic plasma exchange UCB Umbilical cord blood. Frontiers in Immunology 13 frontiersin.org