HLA-haploidentical Family Donors Research Articles

Sequential haplo-identical conditioning transplant regimen for pediatric patients with relapsed or refractory hemophagocytic lymphohistiocytosis.

Allogeneic hematopoietic stem cell transplantation (HSCT) currently stands as the sole remedy for individuals afflicted with hemophagocytic lymphohistiocytosis (HLH). In this study, we retrospectively evaluated how pediatric patients with relapsed or refractory (R/R) HLH responded to our institution's cocktail conditioning regimen. The disease was diagnosed according to criteria applicable to patients with familial/genetic, relapsing, or severe/persistent HLH. All donors were HLA haplo-identical family donors. In our cohort, sixty-five patients (P-HLH), including 28 with familial/genetic HLH, 36 with secondary HLH, and 1 with an unknown cause, underwent haplo-identical family donor HSCT. The conditioning regimen consisted of intravenous administration of etoposide (VP-16), busulfan, fludarabine, rabbit anti-human thymocyte globulin (r-ATG), and cyclophosphamide (Cy). Tacrolimus and mycophenolate mofetil were used for graft-versus-host disease (GvHD) prevention. We observed that the median time for neutrophil recovery was 11 days (range, 8-24), and for platelet counts to exceed 20 × 109/L, it was 14 days (range, 7-130). There were 5 patients (7.7%) who experienced grades III to IV acute GvHD, and 6 patients (9.2%) developed extensive chronic GvHD. The estimated 3- and 5-year overall survival rates were 78.1% (95% CI, 65.8-84.6%) and 74.9% (95% CI, 61.2-84.4%), respectively. The estimated 3- and 5-year event-free survival rates were 73.5% (95% CI, 60.8-82.6%) and 70.3% (95% CI, 56.4-80.5%), respectively. Our findings demonstrate that our innovative conditioning regimen is both effective and safe, offering valuable insights for healthcare professionals evaluating the merits of existing therapies.

HLA-haploidentical hematopoietic stem cells transplantation with regulatory and conventional T-cell adoptive immunotherapy in pediatric patients with very high-risk acute leukemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is still needed for many children with very high-risk acute leukemia. An HLA-haploidentical family donor is a suitable option for those without an HLA-matched donor. Here we present outcomes of a novel HLA-haploidentical HSCT (haplo-HSCT) strategy with adoptive immunotherapy with thymic-derived CD4+CD25+ FoxP3+ regulatory T cells (Tregs) and conventional T cells (Tcons) performed between January 2017 and July 2021 in 20 children with high-risk leukemia. Median age was 14.5 years (range, 4-21), 15 had acute lymphoblastic leukemia, 5 acute myeloid leukemia. The conditioning regimen included total body irradiation (TBI), thiotepa, fludarabine, cyclophosphamide. Grafts contained a megadose of CD34+ cells (mean 12.4 × 106/Kg), Tregs (2 × 106/Kg) and Tcons (0.5-1 × 106/Kg). All patients achieved primary, sustained full-donor engraftment. Only one patient relapsed (5%). The incidence of non-relapse mortality was 15% (3/20 patients). Five/20 patients developed ≥ grade 2 acute Graft versus Host Disease (aGvHD). It resolved in 4 who are alive and disease-free; 1 patient developed chronic GvHD (cGvHD). The probability of GRFS was 60 ± 0.5% (95% CI: 2.1-4.2) (Fig.6), CRFS was 79 ± 0.9% (95% CI: 3.2-4.9) as 16/20 patients are alive and leukemia-free. The median follow-up was 2.1 years (range 0.5 months-5.1 years). This innovative approach was associated with very promising outcomes of HSCT strategy in pediatric patients.

HLA-Haploidentical Family Donors: The New Promise for Childhood Acute Lymphoblastic Leukaemia?

Allogeneic haematopoietic stem cell transplantation (HSCT) is indicated in children with high-risk, relapsed or refractory acute lymphoblastic leukaemia (ALL). HLA-matched grafts from cord blood and stem cell repositories have allowed patients without suitable sibling donors to undergo HSCT. However, challenges in procuring matched unrelated donor (MUD) grafts due to high cost, ethnic disparity and time constraints have led to the exponential rise in the use of stem cells from human leukocyte antigen (HLA)-haploidentical family donors. Whilst HLA-haploidentical HSCT (hHSCT) performed in adult patients with acute leukaemia has produced outcomes similar to MUD transplants, experience in children is limited. Over the last 5 years, more data have emerged on hHSCT in the childhood ALL setting, allowing comparisons with matched donor transplants. The feasibility of hHSCT using adult family donors in childhood ALL may also address the ethical issues related to selection of minor siblings in matched sibling donor transplants. Here, we review hHSCT in paediatric recipients with ALL and highlight the emergence of hHSCT as a promising therapeutic option for patients lacking a suitable matched donor. Recent issues related to conditioning regimens, donor selection and graft-vs.-host disease prophylaxis are discussed. We also identify areas for future research to address transplant-related complications and improve post-transplant disease-free survival.

Haploidentical Natural Killer Cell Therapy As an Adjunct to Stem Cell Transplantation for Refractory Acute Myeloid Leukemia

Refractory acute myeloid leukemia (AML), defined as failure of 2 cycles of induction therapy at diagnosis or of 1 cycle at relapse, represents a subgroup with poor clinical outcomes. In our transplant cohort, the 5-year overall survival in this subgroup was 16% (Ganapule at al. JGO 2017). Haploidentical natural killer cell (NK) therapy is a strategy that is being explored in refractory malignancies. Our in-vitro and animal model data suggest that exposure to arsenic trioxide (ATO) results in enhanced NK cytotoxicity (Alex AA et al. Front. Immunol 2018). Historically, at our center, patients with refractory AML have been treated with cytoreductive therapy (FLAG ± idarubicin or mitoxantrone + etoposide for 3 to 5 days) followed by 1-week rest and then a reduced-intensity transplant with fludarabine + melphalan conditioning while in peak cytopenia. From February 2019, we initiated a phase II single arm clinical trial (CTRI/2019/02/017505) enrolling patients with refractory AML planned for a stem cell transplant in peak cytopenia. Patients received CD56-positive cells from an HLA haploidentical related family donor (other than the stem cell donor, wherever feasible) following cytoreductive chemotherapy. The NK cell donor preference strategy included presence of KIR ligand mismatch, greater number of KIR B motifs (or the B score), lower donor age, and negative donor specific antibodies tested using flowcytometry crossmatch (Figure 1a). CD56-positive selection was done using CliniMACS prodigy system. This was followed by overnight incubation of the CD56 positive cells in autologous plasma with 2 micromolar ATO and 500 U/mL of interleukin-2. The CD56 positive cells were then infused to the patient 1-day after the completion of cytoreductive chemotherapy. This was followed by a reduced intensity stem cell transplant (Figure 1b). The primary outcome variable was 1-year relapse free survival. From February 2019, 14 patients with median age 28 years (IQR: 15.75-31.5) were enrolled in this trial. Six were females. Six had primary-refractory AML while 8 had relapsed-refractory AML. The cytoreductive chemotherapy was FLAG ± idarubicin (n=7), Mitoxantrone + Etoposide (n=6) and GCLAC (n=1). The median blast percentage on flowcytometry MRD testing prior to NK infusion was 15.9% (IQR: 9.1%-54.5%) (n=11). The median B score for the NK cell donors was 2 (IQR: 1-3). The median age of the NK cell donor was 43 years (IQR: 36-49.5). KIR ligand mismatch with the patient was noted in 2 donors. The median CD56-cell dose infused was 46.16 x 10 6/kg (IQR: 25.06-70.36) (Figure 1c). Pre-defined release criteria, including sterile cultures, and endotoxin negativity were met in all cases. There was no infusion related toxicity. The median blast percentage on flowcytometry MRD testing done following NK cell infusion was 11.9% (IQR: 4.9%-47.6%) (n=8). One patient withdrew consent after NK cell infusion and did not undergo transplant. For the remaining 13 patients, the stem cell donor was HLA matched (n=4), HLA 9/10 matched (n=1) or HLA haplomatched (n=8). The median CD34 cell dose infused was 10 x 10 6/kg (IQR:7.51-11.6). Five (38.5%) patients died of immediate post-transplant complications (sepsis (n=3) on days 1, 2 and 28, cerebral venous sinus thrombosis (n=1) on day 1 in a patient treated with hormonal contraceptives for menorrhagia, and veno-occlusive disease (n=1) on day 15 in a patient undergoing a second transplant) while 2 (15.4%) did not engraft (both subsequently died of infective complications following engraftment post-second transplant). Of the remaining 6 (46.2%) patients who engrafted and survived beyond 1 month of the transplant, the day 28 post-transplant MRD was negative for 5 patients while it was positive in 1 patient (0.13%). On follow up, 2 (15.4%) patients developed disease relapse (on days 54 and 218 respectively) and died. The remaining 4 (30.8%) patients are alive and relapse free at last follow up (mean follow up of surviving patients is 16 months). One patient received a CD34 cell boost on day 96 (cell dose - 8.55 x 10 6/kg) for poor graft function. For the entire cohort, the estimated 1-year event free survival is 28.8% ± 13.1%(Figure 1d). Whereas, acute GVHD was noted in 3 patients (50%; out of 6 evaluable patients) and chronic GVHD was noted in 3 patients (50%; out of the 6 evaluable patients). Thus, haploidentical NK cell therapy as an adjunct to transplant is safe and merits further evaluation in patients with AML. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Clinical Outcomes of Microtransplantation for Older Adults with Acute Myeloid Leukemia

Introduction: Outcomes of acute myeloid leukemia (AML) in older patients remain unsatisfactory with low rates of complete remission (CR), disease-free survival (DFS), and overall survival (OS). Microtransplantation - in which allogeneic cells are infused without substantial engraftment (<2% donor cell engraftment) - has the potential to improve anti-tumor immunity without the risks of allogeneic hematopoietic stem cell transplant and graft-vs-host disease (GVHD).Methods: Adapting the protocol of Ai et al. (Blood 2011;117(3):936-41), newly diagnosed high-risk AML patients ≥ 55 years received induction with cytarabine100 mg/m2, idarubicin 12 mg/m2, and microtransplantation (7+3+MST). Microtransplantation was given 24-48 hours after 7+3 and consisted of a G-CSF mobilized leukapheresis product from a HLA-haploidentical family donor; a minimum of 1x108 CD3+ cells/kg were infused. Subjects in CR received 2 cycles of consolidation with high dose cytarabine (HiDAC); the second cycle was followed by another MST. Subjects not in CR were allowed to undergo a 2nd repeat induction cycle (7+3+MST).Results:We enrolled seventeen patients (Table 1) with a median of 248 days of follow up (range 37-887). The median age was 73 years (range 61-78); 5 (29%) had an antecedent hematologic disorder, and 10 (59%) had adverse-risk cytogenetics. Overall, 12 (71%) had poor-risk AML defined as either therapy-related AML, antecedent hematologic disorder, or adverse-risk cytogenetics; 2 (12%) had intermediate-risk AML, and 3 (18%) had favorable-risk AML (all three NPM1 positive).One patient developed sepsis during induction and did not receive microtransplantation; of the other sixteen patients that received at least one round of 7+3+MST, 10 (62.5%) achieved a complete remission, including 4 of 4 patients with intermediate or favorable risk AML (100%) and 6 of 12 with poor-risk AML (50%). Of the 10 in CR, 9 achieved CR after one round of induction and one required a second induction. Nine completed consolidation while one stopped after the first cycle of consolidation due to deconditioning. Three (19%) remain disease-free and alive at 100, 274, and 737 days. Interestingly, all three of these patients had fevers/chills during or immediately after MST. There were no further complications with MST, no cases of treatment-related mortality, and no cases of GVHD, consistent with absence of substantial engraftment. Estimated survival at six months was 67% and at one year was 34% (Figure 1). Correlative studies of immunomodulatory effects, including T-cell exhaustion and NK cell function, depth of response (minimal residual disease), and persistence of donor cells (microchimerism) are pending to better understand the potential mechanisms by which microtransplantation may improve disease responses.Conclusion: Microtransplantation is a potentially safe and effective therapy for older patients with AML. High rates of complete remission were observed in both those with favorable/intermediate risk disease as well as those with poor-risk disease. Further studies are warranted into the efficacy and mechanisms of microtransplantation. [Display omitted] DisclosuresSung:Novartis: Research Funding; Merck: Research Funding; Cellective: Research Funding. LeBlanc:Seattle Genetics: Research Funding; Helsinn Therapeutics: Consultancy, Honoraria; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Pfizer: Consultancy; AstraZeneca: Research Funding; American Cancer Society: Research Funding; Celgene: Honoraria; Cambia Health Foundation: Research Funding; Flatiron Health: Consultancy. Brander:Teva Pharmaceuticals, Genentech, AbbVie, Pharmacyclics: Consultancy; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. McKinney:Kite Pharma: Other: advisory comittee. Sipkins:Gilead Sciences, Inc.: Research Funding. Rizzieri:Shire: Research Funding; Erytech: Research Funding.

Administration of BPX-501 Cells Following Αβ- T and B-Cell-Depleted HLA-Haploidentical HSCT in Children with Fanconi Anemia

Abstract Background Allogeneic hematopoietic stem cell transplantation (HSCT) has been shown to rescue trilineage cytopenias and to prevent occurrence of myeloid malignancies in patients with Fanconi Anemia (FA). HLA-identical family donor HSCT is a recommended treatment strategy with an ≥80% cure rate, and outcomes of HSCT from unrelated donors have progressively improved over time. However, HLA-partially matched (haplo) HSCTs have been historically associated with inferior results. Innovative approaches of graft manipulation, such as selective depletion of αβ-T and B cells, have recently been reported to improve the outcomes of haplo-HSCT in children with non-malignant disorders, however obstacles remain, such as delayed recovery of adaptive immunity with a risk of life-threatening infections. Novel strategies aimed at accelerating immune reconstitution are warranted to optimize the outcomes of patients transplanted from a HLA-haploidentical family donor. BPX-501 is an allogeneic product consisting of T cells modified to express the inducible caspase-9 (iC9) safety switch and truncated CD19 to allow monitoring and expansion of BPX-501 following transplant. The polyclonal nature of the BPX-501 provides broad virus and tumor-specific immunity, while the safety switch provides the unique ability to promptly and durably resolve graft-versus-host disease (GvHD) symptoms following the administration of rimiducid which induces dimerization and activation of iC9, inducing apoptosis of BPX-501. Aims To evaluate the safety and efficacy (acceleration of immune recovery and prevention of transplant-related mortality while maintaining low rates of acute and chronic GvHD) of BPX-501 administered after a αβ T and B-cell depleted haplo-HSCT in pediatric patients with FA. Methods This is a multicenter US (NCT03301168) and EU (NCT02065869), prospective trial utilizing αβ-T- and B-cell-depleted haplo-HSCT followed by infusion of donor lymphocytes genetically modified to express iC9 (BPX-501 T cells). BPX-501 cells were planned to be infused on day14±4 after the allograft per protocol. No post-transplant pharmacological GvHD prophylaxis was employed. Patients who develop GvHD resistant to conventional steroid therapy were eligible to receive ≥1 dose of dimerizing rimiducid activating iC9. The efficacy-evaluable population (EEP) was defined as any patient with FA who received HSCT, BPX-501 infusion and had at least one follow-up assessment. Results At the time of clinical cut-off (June 30th, 2018), 14 patients met the EEP definition. Nine and 5 patients were transplanted in the EU and US, respectively. The median follow-up was 23.5 mos (0.75 - 42 mos). Key baseline and transplant characteristics are detailed in Table 1. The median time for neutrophil and platelet engraftment was 14 (11-15) and 10 (8 - 11) days, respectively. One patient experienced primary graft failure (7.7% [95% CI: 0-22.2%]). Only one patient developed Grade I aGvHD (7.7% [95% CI: 0-22.2%]). Two patients developed cGvHD (19.2% [95% CI: 4-43.2%]), both cases being moderate. Rimiducid was not administered to any patient. One patient died after transplantation due to brain hemorrhage 25 days post HSCT. The event was unrelated to BPX-501 (TRM 7.1% [95% CI: 0-20.6%]). No patients died of infectious complications. The probability of both overall survival (OS) and disease-free survival (DFS) was 92.9% (95% CI: 79.4-100%). The median time from last red blood cell (RBC) transfusion was 23.6 mos (0.2 - 41.9 mos). CD3+ and CD3+CD4+ T cells above 500 cells/ml were achieved by 180 and 360 days, respectively. IgA and IgM levels achieved normal values by 30 and 180 days, respectively. The percentage of circulating and median absolute BPX-501 cells at Day 100 were 3.35% ± 2.94% (0 - 8.4%) and 29.09 ± 39.56 cells/ml (0 - 135 cells/ml), respectively. Conclusion These data indicate that the adoptive transfer of BPX-501 following αβ-T and B-cell depleted haplo-HSCT represents a novel and highly effective transplantation strategy for pediatric patients with FA. Further, despite the addition of BPX-501, low rates and severity of both acute and chronic GvHD was observed, a finding that could translate into significant long-term benefit, in terms of reduction of squamous cell carcinoma a well-known complication occurring in FA patients after HSCT. Disclosures Aldinger: Bellicum Pharmaceuticals, Inc.: Employment. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Steroid-Refractory Acute Gvhd in Children: Retrospective Analysis of the AIEOP HSCT Registry

Introduction. Severe acute graft-versus-host disease (GVHD) remains the most relevant complication after allogeneic HSCT. Although its incidence in the pediatric population is lower than in adults, children with severe acute GVHD and who do not respond to first-line treatment with systemic steroids still have a poor prognosis. The exact incidence of steroid-refractory acute GVHD in children is still not precisely defined, as well as the risk of non-relapse mortality (NRM) due to steroid-refractory acute GVHD. Aim of our study was to analyze the frequency of acute GVHD unresponsive to first-line steroid treatment in children and adolescents given allogenic HSCT, to describe the second line treatment employed, and the outcome of patient with this complication.Patients and methods. This retrospective study included patients younger than 18 years at the time of transplantation and given a first allogeneic HSCT between 2010 and 2015 in one of the HSCT Centers of the Italian Association for Pediatric Hematology / Oncology (AIEOP). Overall, 1608 patients (59% M and 41% F) were analyzed. Median age at HSCT was 8 years (range 0.2 - 18) 1084 (67%) were affected by malignant diseases and 524 (33%) by non-malignant disorders. The donor was an HLA-matched family donor (MFD) in 28% of cases, an unrelated donor (UD) in 52% and an HLA-haploidentical family donor in 20%. In MFD transplants Cyclosporine (CSA) was used as GVHD prophylaxis in 30% of cases and the combination of CSA + short-term methotrexate (MTX) in 48%. 75% of UD transplant recipients received CSA + MTX + anti-thymocyte globulin (ATG) as GVHD prophylaxis, and 25% other drug combinations. Ex vivo T-cell depletion of the graft was employed in most patients given a HLA-haploidentical HSCT (79% of transplants), and high-dose post-transplant cyclophosphamide in 11% of cases.Results. The cumulative incidence (CI) of grade II-IV acute GVHD was 31%, while that of grade III-IV acute GVHD was 10%. The overall incidence of chronic GVHD was 13% and that of extensive chronic GVHD was 6%. The CI of NRM was 14% for grade 0 acute GVHD patients, 9% for grade I, 11% for grade II, 26% for grade III and 68% for grade IV (P < 0.001). Of the 491 patients with grade II-IV acute GVHD, 250 (51%) required a second-line treatment after first-line steroid therapy (30% of grade II, of 75% grade III and 83% of grade IV patients). Acute GVHD requiring second-line treatment was more frequent in UD transplant recipients (21% of patients) than in matched sibling or haploidentical donor recipients (7% and 13% respectively, P < 0.01), while age at HSCT and diagnosis (malignant vs. non-malignant disease) were not associated with this complication. Second-line treatment was extracorporeal photochemotherapy in 60% of patients, mofetil mycophenolate (MMF) in 46%, mesenchymal stromal cells (MSC) in 12%, monoclonal antibodies (MoAbs) in 5% and other treatments in 28%; 32% of patients received more than one second line treatment. Overall NRM was 13% for patients with grade 0-I acute GVHD, 15% for grade II-IV responding to steroids, and 25% for grade II-IV patients requiring second-line therapy (P < 0.001). The addition of a second-line treatment partially decreased NRM only in patients with grade IV acute GVHD, but the difference was not statistically significant (66% vs. 78%; P = 0.313). In multivariable analysis, grade III (HR = 1.84; P = 0.044) and grade IV acute GVHD (HR = 7.07; P < 0.001), and the use of an UD (HR = 1.63; P = 0.007) were associated with an increased NRM, while the use of a second-line treatment did not decrease this risk (HR = 0.80; P = 0.368).Conclusions. Despite being less frequent than in adults, severe steroid-refractory acute GVHD is still associated with a very high NRM also in pediatric patients. Second or third-line treatments adopted so far have not been effective in improving control of the complication and in decreasing NRM. The prospective evaluation of acute GVHD biomarkers (such as ST2 and REG3α) could help in identifying patients at higher risk of NRM. Prospective studies are warranted to define new treatment modalities that could decrease the mortality rate associated with the most severe form of disease. DisclosuresZecca:Chimerix: Honoraria. Locatelli:bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

Mutational Landscape of Pediatric Acute Lymphoblastic Leukemia Relapsing after Allogeneic Stem Cell Transplantation

The prognosis of children with acute lymphoblastic leukemia (ALL) relapsing following allogeneic hematopoietic stem cell transplantation (allo-SCT) is still dismal. Within the framework of the ALL-REZ BFM 2002/ALL-SZT BFM 2003 trials, we performed whole-exome sequencing (WES) of ten patients relapsing after allo-SCT with the aim to thoroughly characterize the spectrum of acquired mutations and to identify potentially druggable targets, thus laying the ground for future personalized treatment strategies.Patients’ age at initial diagnosis ranged from <12 months to 10 years. Two patients were diagnosed with T-ALL and 8 with pre-B-ALL. Time from initial disease to relapse ranged from 13 to 40 months. Patients underwent allo-SCT 3 to 6 months after diagnosis of relapse, donors were either HLA-matched unrelated volunteers (n=6), HLA-identical siblings (n=1) or HLA-haploidentical family donors (n=1). Time from SCT to relapse ranged from 3 to 21 months. Treatment following the post allo-SCT relapse varied considerably (from palliative care to a second allo-SCT). Only 2/10 patients are still alive, with one recently having been diagnosed with subsequent relapse following a second SCT.To investigate the mutational landscape of relapsed ALL following high-dose chemotherapeutic and immunologic attack both provided by an allo-SCT, five samples per patient were analyzed: initial leukemia (INIT), remission (REMI) after front-line chemotherapy representing patient germline, first relapse (RLPS), full donor-chimeric remission post allo-SCT (TREMI) representing donor germline, and relapse post allo-SCT (TRLPS). For comparative analyses, we defined the following three "oncogenomes" (OGs): OG1 (initial leukemia, SNVs in INIT minus REMI)), OG2 (first relapse, SNVs in RLPS minus REMI), OG3 (post allo-SCT relapse, SNVs in TRPLS minus REMI or TREMI).Median numbers of leukemia-specific SNVs in OG1-3 were 8.5, 34 and 37.5. We detected a median of 0.18 mutations per megabase (MB) in OG1, 0.67 mutations/MB in OG2 and 0.74 mutations/MB in OG3 (p= 0.005 for OG2/3 vs. OG1 [Wilcoxon test]).Although the mutational spectrum was highly diverse between individual patients and within the oncogenomes, we also identified several recurrent alterations: in OG1, five genes had recurrent SNVs (IGSF3, TTN, NOTCH1, CTBP2, NRAS). Seven genes were recurrently affected in three OG2s, including IKZF1, NOTCH1, NRAS, NT5C2. In OG3 we detected eleven recurrently mutated genes in three patients, among which were NRAS, FLT4, and TP53.Notably, TP53 was mutated in 5/10 patients. One patient carried a TP53 germline mutation, one patient had one unique SNV each in OG2 as well as OG3 and three patients had TP53 mutations only present in their OG3s. All but one SNV (resulting in a premature stop codon) were non-synonymously coding and were predicted to be deleterious for protein function.Of particular note, leukemic blasts showed profound plasticity concerning the mutational status of the nucleoside exporter NT5C2, mutations of which were previously shown to drive chemotherapy resistance in relapsed childhood ALL. While NT5C2 alterations were completely absent in OG1, 4 SNVs were detected in the OG2s of 3/10 patients. However, these NT5C2 mutations disappeared again in the OG3s once selection pressure of maintenance chemotherapy employing nucleoside analogues had been withdrawn.To identify novel treatment options in the desperate clinical scenario of post allo-SCT relapse, we searched OG3 for genetic lesions in genes known to be either targeted directly (a certain gene) or indirectly (a certain pathway) by currently approved therapeutic agents. To our surprise, nine out of ten patients exhibited such SNVs, for which additional targeted therapies are already available. Those therapeutic agents comprise small molecules inhibitors as well as antibodies such as Dasatinib, Erlotinib, Ibrutinib, Pazopanib, Tocilizumab, and Trastuzumab.Conclusion:Our comprehensive genetic analysis of ten children with ALL relapsing post allo-SCT revealed profound leukemic cell plasticity (e.g. loss of acquired NT5C2 mutations) as well as identified several recurrent genetic alterations (e.g. NOTCH1, NRAS, IKZF1) including TP53 (5/10 patients). Most importantly, we identified alterations in genes amenable to targeted treatment approaches in 9/10 patients thus potentially opening new therapeutic avenues. DisclosuresBader:Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Neovii Biotech: Research Funding; Riemser: Research Funding; Medac: Consultancy, Research Funding. Peters:Novartis: Consultancy; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy; Medac: Consultancy.

Mother Donors Improve Outcomes after HLA Haploidentical Hematopoietic Transplantation: A Retrospective Study By the Cell Therapy and Immunobiology Working Party of the EBMT

▪IntroductionTrans-placental trafficking of maternal and fetal cells during pregnancy establishes long-term, reciprocal micro-chimerism in both mother and child (Maloney et al., J Clin Invest, 104:41, 1999). As a consequence, the immune system of the mother may become sensitized to paternal histocompatibility antigens. In fact, antibodies directed against paternal HLA-antigens (van Rood JJ et al., Nature 181:1735, 1958) and T lymphocytes directed against paternal major and minor histocompatibility antigens (van Kampen CA et al., Hum Immunol 62:201, 2001; Verdijk RM et al., Blood 103:1961, 2004) were detected in multiparous women. More recently, it was hypothesized that mother's "exposure" to paternal HLA haplotype antigens during pregnancy may affect transplantation outcomes when the mother acts as donor for the child. Indeed, survival after T cell-depleted HLA haploidentical haematopoietic transplantation was improved using the mother as donor (vs all other family members) (Stern et al., Blood 112:2990, 2008; Kruchen et al., BMT 50:1367 2015). However, maternal donors were associated with increased incidence of GvHD and decreased survival after un-manipulated HLA haploidentical blood and marrow grafts (Wang Y et al., Blood 124:843, 2014).Patients and MethodsA retrospective EBMT registry-based study was performed in a combined series of adult (n=333) and pediatric (n=105) patients with acute leukemia (AML=268, ALL=160, Mixed phenotype=10) who received transplant from one MHC mismatched family donor. Forty-four percent (233) of patients were in I or II complete remission (CR) at the time of transplant, 193 were in chemo-resistant relapse or in CR > 2. Median age was 32.7 (range: 0.67-70). Seventy-one percent of patients received ex-vivo T cell depleted transplants. Twenty-two patients were given bone marrow and peripheral blood stem cells, the others were given peripheral blood cells only. The mother was used as donor in 98 patients.ResultsEighty patients developed acute GvHD ≥ grade II. At a median follow up of 58.7 months, 130 patients are alive without disease, 146 relapsed and 152 died of non-relapse mortality (NRM). Univariate analyses of factors influencing relapse-free survival (RFS) showed age, disease status at transplant, Karnofsky score and ex-vivo T cell depletion impacted significantly. As transplantation outcomes from family members other than mothers did not differ from one another, such transplants were combined for analyses. When compared with transplantation from all other family members (n=330), transplantation from mother donors (n=98) was associated with lower relapse incidence (RI) (28% vs 39%, P=0.06) and non-relapse mortality (NRM) (30% vs 40%, P=0.09) and, consequently, better relapse-free survival (RFS) (43% vs 21%, P < 0.001). Multivariate analyses showed transplantation from mother donors was an independent factor predicting better RFS (other donors vs mothers: HR: 1.42; CI: 1.01-2.00; P=0.04) and lower RI (other donors vs mothers: HR: 1.93; CI: 1.16-3.22; P = 0.01). In addition, transplantation in relapse (vs remission) predicted worse RFS (HR: 2.33, CI: 1.80-3.00, P < 0.001) such as ≥18 (vs < 18) adversely impacted on RFS (HR: 1.40, CI: 1.00-1.96, P=0.04).DiscussionOur retrospective analyses in 438 HLA haploidentical hematopoietic transplants for acute leukemia patients (pediatric and adult) show that transplantation from mother donors, when compared with transplantation from any other family member, is an independent factor predicting better outcomes, i.e., better RFS and lower RI. Mothers should therefore be preferred when selecting an HLA haploidentical family donor. Further clinical and preclinical studies are needed to unveil the mysteries underlying mother-to-child immune interaction during pregnancy and its bearing on the reproductive success of the human species. DisclosuresCiceri:MolMed SpA: Consultancy. Bonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy.

Selective Depletion of αβ T Cells and B Cells for Human Leukocyte Antigen–Haploidentical Hematopoietic Stem Cell Transplantation. A Three-Year Follow-Up of Procedure Efficiency

HLA-haploidentical family donors represent a valuable option for children requiring allogeneic hematopoietic stem cell transplantation (HSCT). Because graft-versus-host diseases (GVHD) is a major complication of HLA-haploidentical HSCT because of alloreactive T cells in the graft, different methods have been used for ex vivo T cell depletion. Removal of donor αβ T cells, the subset responsible for GVHD, and of B cells, responsible for post-transplantation lymphoproliferative disorders, have been recently developed for HLA-haploidentical HSCT. This manipulation preserves, in addition to CD34+ progenitors, natural killer, γδ T, and monocytes/dendritic cells, contributing to anti-leukemia activity and protection against infections. We analyzed depletion efficiency and cell yield in 200 procedures performed in the last 3 years at our center. Donors underwent CD34+ hematopoietic stem cell (HSC) peripheral blood mobilization with granulocyte colony–stimulating factor (G-CSF). Poor CD34+ cell mobilizers (48 of 189, 25%) received plerixafor in addition to G-CSF. Aphereses containing a median of 52.5 × 109 nucleated cells and 494 × 106 CD34+ HSC were manipulated using the CliniMACS device. In comparison to the initial product, αβ T cell depletion produced a median 4.1-log reduction (range, 3.1 to 5.5) and B cell depletion led to a median 3.4-log reduction (range, 2.0 to 4.7). Graft products contained a median of 18.5 × 106 CD34+ HSC/kg recipient body weight, with median values of residual αβ T cells and B cells of 29 × 103/kg and 33 × 103/kg, respectively. Depletion efficiency monitored at 6-month intervals demonstrated steady performance, while improved recovery of CD34+ cells was observed after the first year (P = .0005). These data indicate that αβ T cell and B cell depletion of HSC grafts from HLA-haploidentical donors was efficient and reproducible.

Allogeneic hematopoietic stem-cell transplantation for adult and adolescent hemophagocytic lymphohistiocytosis: a single center analysis.

Myeloablative conditioning-based allogeneic hematopoietic stem-cell transplantation (allo-HSCT) in the treatment of adult and adolescent hemophagocytic lymphohistiocytosis (HLH) is rarely reported. We conducted a retrospective study of 30 adult and adolescent HLH transplanted for primary HLH (n=4), tumor-HLH (n=8), EBV-HLH (n=14), and underlying disease-unknown (UDU)-HLH (n=4). Peripheral blood stem cells (PBSCs) were the stem-cell source in all patients. Twenty-three patients were transplanted from HLA-haploidentical family donors, six from HLA-identical sibling donors, and one from a matched unrelated donor. Four patients appeared with mixed chimerism (MC), and no patient presented with graft failure. There was a high risk for EBV reactivation with an incidence of 47%. Two patients developed post-transplant lymphoproliferative disorder (PTLD) and three were considered primary disease recurrent. With a median follow-up of 26months, 19 patients survived and 11 patients died. The estimated 2-year overall survival (OS) was 63.3±8.8% in all patients, 100% in primary HLH, 64.3±12.8% in EBV-HLH, 50.0±17.7% in tumor-HLH, and 50.0±25.0% in UDU-HLH. Myeloablative conditioning-based allo-HSCT is an effective treatment for adult and adolescent HLH to achieve complete remission and long-term survival.

From immunomonitoring to immune intervention.

Host immune status is a key issue in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the long-term follow-up of these patients, severe post-transplant infections, relapse or secondary malignancies may be directly related to prolonged immune defects, especially in the context of innovative stem cell sources such as umbilical cord blood transplantation (CBT) (1) or transplants from HLA haplo-identical family donors (Haplo-HSCT) (2, 3). This Frontiers Research Topic provides insights into mechanisms of immune reconstitution in an allogeneic environment, in order to improve monitoring and therapeutic intervention in allo-HSCT patients. Forcina et al. (4) critically review T and NK biomarkers, their threshold and clinical relevance. Many factors from the host, transplant conditioning, stem cell source, and genetic disparity may impact immune recovery. Few biomarkers have reached a clinical consensus: a rapid and potent CD4 T-cell recovery is associated with a favorable clinical outcome, CMV-specific CD8 T-cell counts measured by tetramers can predict a lower incidence of CMV disease. Other phenotypic or molecular markers, still based on small-sized studies, will need further validation in large scale-multicentric cohorts. This is the case of molecular markers of lymphocyte generation, such as T-cells evaluated by the quantification of T-cell rearrangement excision circles (TREC) as a surrogate marker of thymic activity. Clave et al. (3) indicate an impact of thymic function recovery on relapse following CBT as well as Haplo-HSCT in children treated for an hematological malignancy. In order to reach the routine medical practice, immunomonitoring tests need also to be simple and fast. This practical issue is well taken into account by Fuji et al. (5) reviewing T-cell monitoring of viral and fungal infections. CMV is still the most intensively studied virus in immunocompromised hosts. CMV shapes T and NK cell responses in many ways and may escape immune response. Among still unanswered questions, is the role of viral-specific (CMV and EBV) T-cell cross-reactivity against allogeneic targets in graft-versus-host and graft-versus-leukemia (6). Experimental models have proved their importance in allo-HSCT, especially deciphering mechanisms of allogeneicity in graft-versus-host disease (GVHD). Among rodents, rat models are relevant especially in some autoimmune conditions and solid organ transplantation, sometimes closer to the human disease than the “gold standard” murine models. Zinocker et al. (7) emphasize the interest of rat model in experimental GVHD, especially the rat skin explant assay as a tool for functional evaluation of GVHD. Experimental models are also required to provide the basis and for preclinical evaluation of immune-based therapies. This Frontiers Research Topic also reviews in some strategies already proposed in the clinics. Adoptive T-cell therapies have been conducted successfully by several groups to control life-threatening viral (EBV, CMV) reactivations. This leads to “off the shelf” strategies based on third party HLA-typed T-cell donors registries, which may have a major impact in transplant recipients (8). The development of therapeutic antibodies in cancer is one of the most active fields in clinical immunology. Many strategies are underway to improve their action, especially through their antibody-dependent cellular toxicity mediated by natural killer (NK) cells (9). One approach especially relevant in allo-HSCT would be to take advantage of the killer-immunoglobulin-like receptor (KIR) ligand incompatibility in addition to ADCC to boost NK functions. The impact of these different concepts, from basic knowledge to translational medicine, is integrated in state-of-the art reviews of two main “success stories” in allo-HSCT: CBT (1) and Haplo-HSCT (2).

Unmanipulated Haploidentical Transplants Compared with Other Alternative Donors and Matched Sibling Grafts

We studied 459 consecutive patients with hematologic malignancies, median age 44 years (range, 15 to 71 years), who underwent transplantation with grafts from identical sibling donors (SIB; n = 176), matched unrelated donors (MUD; n = 43), mismatched unrelated donors (mmUD; n = 43), unrelated cord blood (UCB; n = 105) or HLA-haploidentical family donors (HAPLO; n = 92). Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate in the SIB recipients; antithymocyte globulin for the MUD, mmUD, and UCB recipients; and post-transplantation cyclophosphamide, cyclosporine, and mycophenolate in the HAPLO recipients. Conditioning regimens were mostly myeloablative (69%). Advanced disease phase was more frequent, but not significantly so, in the HAPLO and mmUD groups (P = .08). Acute GVHD grade II-IV was significantly less frequent in the HAPLO, UCB, and MUD groups (14% to 21%) compared with the SIB (31%) and mmUD (42%) groups (P < .001), and there was a trend toward less moderate-severe chronic GVHD in the HAPLO and UCB groups (P = .053). The proportion of patients off cyclosporine at 1 year ranged from 55% for the SIB group to 81% for the HAPLO group (P < .001). Transplantation-related mortality at 2 years was lower in the HAPLO and SIB groups (18% to 24%) compared with the MUD, mmUD, and UCB groups (33% to 35%; P = .10). Relapse rate was comparable in the 5 groups (P = .80). The 4-year actuarial survival was 45% in the SIB group, 43% in the MUD group, 40% in the mmUD group, 34% in the UCB group, and 52% in the HAPLO group (P = .10). In multivariate analysis, advanced disease was a negative predictor of survival (hazard ratio [HR], 2.4; P < .0001), together with a diagnosis of acute leukemia (HR, 1.8; P = .0001); HAPLO grafts were comparable to SIB (P = .80), whereas UCB had inferior survival (P = .03). In conclusion, unmanipulated haploidentical family donor transplants are an additional option for patients lacking a matched sibling donor.

T-cell replete haploidentical donor transplantation using post-transplant CY: an emerging standard-of-care option for patients who lack an HLA-identical sibling donor.

Availability of an HLA-identical sibling (MRD) or suitably matched unrelated donor (MUD) has historically been a limiting factor in the application of allogeneic hematopoietic transplantation. Although almost all patients have an HLA-haploidentical family donor, prior attempts at transplantation from such donors using T-cell replete grafts and conventional immunosuppression were associated with unacceptable rates of GVHD, and when stringent ex vivo T-cell depletion was used to control GVHD, rates of graft rejection and post-transplant infections were prohibitive. The recent approach to HLA-haploidentical donor transplantation developed in Baltimore that uses T-cell replete grafts and post-transplant CY (Haplo-post-HCT-CY) to control post-transplant allo-reactivity appears to have overcome many of the obstacles historically associated with haploidentical donor transplantation. In particular, TRM rates of <10% are usual and rapid reconstitution of immunity leads to a low rate of post-transplant infections and no post-tranplant lymphoproliferative disorders (PTLD), consistent with the hypothesis that post-transplant CY selectively depletes proliferating alloreactive T cells responsible for GVHD and graft rejection while preserving resting memory T cells essential for post-transplant immunologic recovery. In parallel trials using similar non-myeloablative conditioning regimens, Haplo-post-HCT-CY produced similar overall survival to double umbilical cord blood transplantation(DUCBT) in adult patients (62% vs 54%), with low rates of TRM (7% vs 24%), severe acute GVHD (0% vs 21%) and chronic GVHD (13% vs 25%). Furthermore, recent non-randomized comparisons adjusted for risk factors show that Haplo-post-HCT-CY achieve at least equivalent outcomes to conventional MRD and MUD transplants. Although most experience has been obtained using BM, emerging data suggest that a G-CSF mobilized PBSC graft can also safely be used for Haplo-post-HCT-CY. Haplo-post-HCT-CY also avoids the graft acquisition costs of DUCBT and MUDs and the cost of cell selection associated with T-depleted grafts. Although randomized comparisons will be forthcoming, Haplo-post-HCT-CY can already be considered a valid standard-of-care in patients who lack conventional donors thus extending the availability of allogeneic transplants to almost all patients. This donor source may also challenge the routine preference for a MUD in patients lacking an MRD.

Beneficial Effects of Pretransplantation Microchimerism on Rejection-Free Survival in HLA-Haploidentical Family Donor Renal Transplantation

Fetal-maternal microchimerism (MC) can develop during pregnancy and may persist for decades. Pretransplantation fetal-maternal MC may be present between mother and child and between siblings; however, its effect on renal transplantation is not known. We investigated the effects of pretransplantation MC on allograft outcomes in human leukocyte antigen (HLA)-haploidentical family donor transplantation. A total of 106 cases transplanted from 1996 to 2004 were retrospectively studied, with median follow-up of 96 months. The study and control groups included 63 and 43 cases of HLA-haploidentical and HLA-identical donor transplantations, respectively. MC against mismatched donor HLA-DRB1 allele was detected in the recipient's peripheral blood using nested polymerase chain reaction-single-strand conformation polymorphism method. The allograft outcomes of HLA-haploidentical MC (+) and (-) subgroups were compared with those of HLA-identical group. Pretransplantation MC in the HLA-haploidentical recipients was detected in 22.2% (14 of 63). Compared with HLA-identical group, MC (-) subgroup showed significantly inferior allograft outcomes: higher acute rejection rate (11.6% vs. 42.9%; P=0.001), higher 5-year serum creatinine level (1.1 vs. 1.4 mg/dL; P=0.009), and lower 10-year rejection-free survival rate (83.7% vs. 54.5%; log-rank P=0.001). In contrast, MC (+) subgroup showed no significant differences from HLA-identical group in acute rejection rate (14.3%), 5-year serum creatinine level (1.1 mg/dL), and 10-year rejection-free survival rate (85.7%). Multivariate analysis revealed that pretransplantation MC is associated with a significantly lower risk of acute rejection (odds ratio=0.10; P=0.021). Pretransplantation MC present in the recipient may have beneficial effects on rejection-free allograft survival in renal transplantation.

Thymic function recovery after unrelated donor cord blood or T-cell depleted HLA-haploidentical stem cell transplantation correlates with leukemia relapse

Use of alternative donors/sources of hematopoietic stem cells (HSC), such as cord blood (CB) or HLA-haploidentical (Haplo)-related donors, is associated with a significant delay in immune reconstitution after transplantation. Long-term T-cell immune reconstitution largely relies on the generation of new T cells in the recipient thymus, which can be evaluated through signal joint (sj) and beta T-cell-Receptor Excision Circles (TREC) quantification. We studied two groups of 33 and 24 children receiving, respectively, HSC Transplantation (HSCT) from an HLA-haploidentical family donor or an unrelated CB donor, for both malignant (46) and non-malignant disorders (11). Relative and absolute sj and beta-TREC values indicated comparable thymic function reconstitution at 3 and 6 months after the allograft in both groups. Compared to children with non-malignant disorders, those with hematological malignancies had significantly lower pre-transplantation TREC counts. Patients who relapsed after HSCT had a significantly less efficient thymic function both before and 6 months after HSCT with especially low beta-TREC values, this finding suggesting an impact of early intra-thymic T-cell differentiation on the occurrence of leukemia relapse.

Negative Depletion of B Cells and T Cells Expressing the αβ Chain of the T-Cell Receptor (TCR) for Haploidentical Stem Cell Transplantation

Abstract 343 Introduction.Allogeneic hematopoietic stem cell transplantation (HSCT) from a HLA-haploidentical relative is a suitable option for patients (pts) lacking a compatible donor, either related or unrelated. The two main approaches for overcoming the obstacles of HLA barriers are based either on the infusion of large numbers of T-cell-depleted HSC or on intensive pharmacological prevention of graft-versus-host disease (GVHD). While for many years T-cell depletion (TCD) of the graft has been based on either immunomagnetic positive selection of CD34+ cells or on physical removal of all subsets of T cells by virtue of mAb, we and other groups have recently developed a novel method of ex vivo TCD based on the selective elimination of αβ+ T cells through labeling with a biotinylated anti-TCRαβ Ab, followed by incubation with an anti-biotin Ab conjugated to paramagnetic beads (Miltenyi Biotec, Germany). This approach also allows the removal of B cells to prevent post-transplant EBV-associated lymphoproliferative disease (PTLD). Here, we report the results of graft manipulation using this approach. Methods.Twenty-two children entered the study, 15 with hematological malignancies and 7 with non-malignant disorders. No post-transplant GvHD prophylaxis was employed. HLA-haploidentical family donors received G-CSF (12–16 μg/kg of body weight) to mobilize HSC prior to large-volume leukapheresis, which was commenced when circulating CD34+ HSC were >20 cells/μl. Cell therapy products containing up to 60×109 white blood cells (WBC) were processed according to the manufacturer’s protocol. In some cases, leukapheresis bags were stored overnight at 4°C in appropriate media at a WBC concentration <200×106/ml. Aliquots of the graft were used to enumerate residual αβ T cells and B cells, as well as other immune effector cells, including type 1 and 2 dendritic cell (DC) precursors, invariant NKT cells, classical CD14+CD16− and non-classical CD14+CD16+ monocytes. All TCR αβ+/CD19+cell-depleted grafts were re-infused within 2 hours from the end of processing. Results.Median recovery of CD34+ HSC and median number of infused CD34+ HSC were 99.3% (range 55.4–100) and 14.7×106/kg (range 7.9–37), respectively. The graft contained 3.9×106 CD3+ T cells/kg (range 0.9–30.9) and 0.08×106 B cells/kg (range 0.002–0.32). The log-depletion of αβ+ T cells was 4.5 (range 3.2–6.1), with a median number of transplanted αβ+ T cells equal to 36×103/kg (range 1–139.9). Patients received 36×106 CD56+ NK cells/kg (range 1.6–80.3) and 4.1×106 γδ+ T cells/kg (range 0.9–30.8). A median of 40.8% NK cells (range 30.3–60.2) co-expressed Tim-3, an activating co-receptor promoting IFN-γ production and being scarcely expressed on NK cells that reconstitute after allogeneic HSCT. The αβ/CD19-depleted grafts also contained DC1 and DC2 precursors, which were identified based on BDCA-1 (0.23% of all nucleated cells, range 0.02–2.4), BDCA-3 (0.93%, range 0.57–9.4) and BDCA-2 (0.66%, range 0.49–1.28) or BDCA-4 expression (0.69%, range 0.02–1.5), respectively. Invariant NKT cells were enumerated using mAb reacting against the TCR Vα24-Jα18 chain and were detected in minute percentages (<0.05% of all nucleated cells) within the graft. The αβ/CD19-depleted grafts were also enriched in non-classical CD14+CD16+ monocytes (13.7% of all CD14+cells, range 9.3–29.4), which have been reported to predict a reduced incidence of GVHD after allogeneic HSCT. All patients rapidly engrafted, the median time to reach 500 neutrophils and 50,000 platelets per μl of blood being 12 days (range 10–16) and 13 days (range 12–18), respectively. Only 2 pts developed skin grade I/II acute GVHD, while no pt had visceral acute GVHD. With a median follow-up of 6 months (range 2–8), no pt experienced EBV-related PTLD or died of transplant-related complications. Conclusions.The immunomagnetic removal of αβ+ T cells and B cells was effective and gave reproducible results. In all cases, a remarkable depletion of unwanted T and B cells was attained. The depleted graft contained high numbers of CD34+ HSC, as well as of immune effector cells implicated in the control of leukemia growth and GVHD, such as γδ+ T cells, NK cells, DC1, DC2 and non-classical CD14+CD16+ monocytes. From a clinical standpoint, the infusion of αβ/CD19-depleted grafts from HLA-haploidentical family donors was safe, resulting into sustained donor engraftment without life-threatening complications. Disclosures:No relevant conflicts of interest to declare.

LOW Tranplant Related Mortality and LOW GRAFT Rejection After Related Haploidentical STEM CELL Transplantation (HAPLO-SCT) Prepared with NON Myeloablative CONDITIONING REGIMEN (NMA) and Posttransplant Cyclophosphamide for Advanced Lymphoid Malignancies.

AbstractAbstract 3031 Introduction:The use of hematopoietic stem cells from partially matched family donors has become an alternative transplant option for patients with no HLA identical donor available. It might be even a first choice, especially in advanced hematological maligancies, because of the presumed stronger graft versus tumor effect relied to the partial HLA identity. However, haplo-SCT is associated with high risk of graft rejection (GR) and severe graft versus host disease (GvHD), especially after non NMA. High dose posttransplant Cyclophosphamide (Cy) has been shown to prevent GvHD and GR. Aim:This retrospective analysis report the outcome of 15 patients (pt) using posttransplant Cy after a NMA in our center. Results:Fifteen adult pt (Male=9) with high risk lymphoid malignancies for whom a HLA matched related or unrelated donor were not available, underwent haplo-SCT from February 2011 to June 2012. The median pt age was 41years (y) (range, 20–60). Diagnosis were Hodgkin Lymphoma in 5 pt, Non Hodgkin Lymphoma in 4 pt, Chronic Lymphatic Leukemia in 2 pt, Multiple Myeloma in 2 pt and Acute Lymphoblastic Leukemia in 3 pt. All pt were heavily pretreated with a median of 3 or more chemotherapy regimens including failure of previous autologous (12pt) or allogeneic transplantation (3pt). At time of transplant 9pt (60%) were in complete remission (CR1=6, CR2=3). The NMA consisted in Fludarabine 30mg/m2 iv on days -6 to -2, Cyclophospamide 14.5mg/kg iv from days -6 and -5 and Total Body Irradiation 200cGy on day -1. For Graft versus Host Disease prophylaxis all pt received postransplant Cy 50mg/kg iv on days + 3 and +4 with Mesna. One day later, all pt started with Cyclosporine (CSA) 3mg/kg/day and Mycophenolatemofetil (MMF) 15mg/kg/day and Filgrastim 5ug/kg/day until neutrophil recovery. MMF was stopped at day+35 and CSA was given until day +180 in the absence of GvHD. Ten pt (67%) received bone marrow (BM) and 5 pt (33%) G-CSF mobilized peripheral blood stem cells (PBSC) as graft source. All donors were HLA haploidentical first degree family donors (Male=8) including brothers (7), sisters (2), mothers (4) and son (1). Median donor age was 49 y (range, 23–66). Pt had HLA typing at the allele or antigen level (A, B, Cw, DRB1, DQ) whereas 8 pt were mismatched at 5 loci, 1 pt at 4 loci, 4 pt at 3 and 2 pt at 2 loci. BM grafts contained a medium of 2×106/kg CD34+ cells and 22×106/kg CD3+ cells. PBSC grafts contained a medium of 6×106/kg CD34+ cells and 298×106/kg CD3+ cells. The engraftment rate was 93%, with a median time to neutrophil recovery (>500 G/l) of 20.5 days (range, 14–26) and to platelet recovery (>20 000 G/l) of 28.5 days (range, 14–41). Compared to BM recipients, pt receiving PBSC experienced faster neutrophil [(17 days (range, 14–23) vs 21 days (range, 18–26), p=0.04] and platelet recovery [(23 days (range, 14–30) vs 30 days (range, 23–41), p=0.04)]. One pt with ALL did not engraft and died at day +40 for infectious complication. One pt died of treatment related mortality with a cumulative incidence of 7% [96%CI (0–20)]. Two pt developped acute GvHD grade 2. No chronic GvHD was observed in pt surviving beyond day 100. Four pt exerienced relapse or progression of the underlying disease. After a median follow up of 197 days (range, 40–518) the actuarial overall survival and event-free survival at 2 years were 87% [95% CI (70–100)] and 51 % [95% CI (16–85)], respectively. Conclusion:NMA haplo-SCT with postransplant Cy in pt with advanced lymphoid malignancies is associated with low NRM, low acute GvHD and low graft rejection. However, better disease control in selected pt is needed to overcome early relapse or progression. Even if follow-up is short, PBSC seems to be an promising option to bone marrow as stem cell source. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Haploidentical hematopoietic stem cell transplantation for lymphoma with monosomy of chromosome 6 (loss of heterozygosity in the HLA region) — Who should be a donor?

Hematopoietic stem cell transplantation (HSCT) from an HLA haploidentical family donor is an option for patients who do not have a full HLA matched donor and lack the time to find an unrelated one [1,2]. Furthermore, it may facilitate a powerful graft versus leukemia/lymphoma (GVL) effect to help combat hematological malignancies by directly targeting the mismatched HLA expressed on leukemia/lymphoma cells [3]. On the contrary, leukemia/lymphoma cells escape from the surveillance of the donor-derived GVL effect by losing the target HLA (mismatched HLA in GVH direction). This mechanism has been called loss of heterozygosity (LOH) in the HLA gene region on chromosome 6 [4,5]. Taking the above into account, in this case report, we present a case of lymphoma with monosomy 6, which means natural LOH of HLA, and suggest that selection of a haploidentical family donor matched with the missing HLA haplotype seems to be very effective.

Increase In NK Cell Lysis of Leukemic Blasts Due to Loss of Mismatched HLA Haplotype After Haplo-Identical Stem Cell Transplantation

AbstractAbstract 2540 Introduction:Hematopoietic stem cell transplantation (HSCT) from HLA haplo-identical family donors is promising as a therapy for patients with leukemia who are at high risk for relapse. The lower relapse rates and improved survival, especially for patients with acute myelogenous leukemia (AML) who have received HSCT from killer cell immunoglobulin-like receptor (KIR) ligand-mismatched donors, suggest that donor NK cells that are alloreactive against the recipient's cells cause graft-versus-leukemia effects. An Italian group and we identified genomic loss of the patient-specific HLA haplotype in leukemic cells after haplo-identical HSCT. Analysis using SNP arrays revealed that the HLA loss in 29 to 66% of relapsed patients after haplo-identical HSCT was caused by segmental uniparental disomy (UPD) of the HLA region on chromosome 6. This suggested that leukemic cells often escape immunosurveillance through the loss of the mismatched HLA haplotype via the UDP mechanism after haplo-identical HSCT (Vago et al. N Engl J Med. 2009, Villalobos, IB et al. Blood 2010). Since NK cell effector function is tightly regulated by inhibitory KIRs on NK cells that bind to MHC class I on target cells, the escape of leukemic cells from immune surveillance by losing a mismatched HLA antigen might enhance the cytotoxicity of NK cells towards target cells. We examined alterations in donor-derived alloreactive NK cell activity against leukemic blasts of AML patients who relapsed after HLA haplo-identical HSCT. Patients and methods:We enrolled three patients with AML, aged 2, 3 and 12 years, who relapsed after HLA haplo-identical HSCT with T cell depletion of rabbit ATG in vivo. Two patients had AML M7 and the other had M0. Only one donor was KIR ligand-mismatched. Engraftment was achieved in all three patients within 28 days. Relapse occurred 35, 372 and 445 days after HSCT. Mononuclear cells were obtained from both donors and patients before and after HSCT. The NK cells were purified using NK cell selection kits (DYNAL) and measured by conventional 51Cr release assays of leukemic blasts from the patients and the control cell line K562. The mismatched HLA expression between patients and donors on hematopoietic cells was monitored by flow cytometry using anti-HLA antibody (One Lambda). Results:The mean of NK cell killing of the patients' leukemic cells at the same effector target ratio of 10 was significantly higher in donors (50.3%) than in relapsed patients (9.7%). Monitoring of alloreactive NK cell activity after HSCT showed that donor-derived NK cell killing against the patients' leukemic cells gradually decreased in the KIR ligand-mismatched setting. Relapse was minimal but obvious according to HLA monitoring of hematopoietic cells on day 98, which was 7 days after alloreactive NK cell activity was diminished in the patients. Monitoring HLA expression after HSCT also revealed that one patient had leukemic blasts at relapse with loss of the patient's specific HLA haplotype caused by UDP of the HLA region on chromosome 6. Notably, primary leukemic blasts in this patient were not killed by the donor NK cells (2.3%), but leukemic blasts at relapse were efficiently killed (69.0%) after HLA loss of leukemic cells. On the other hand, killing of leukemic blasts at relapse by the patient's NK cells after transplantation was much less effective (19.0%) than that by donor NK cells, although they originated from the same donor (Figure). Because haplotype loss of HLA caused by UDP does not change the status of the KIR ligand in patients with homozygous HLA-Cw, we further examined the expression of ULBP1-3 that are ligands for the activating NK receptor, NKG2D, on leukemic blasts before and after HLA loss. We found upregulated ULBP-2 expression on leukemic cells after the loss of HLA. Conclusions:Donor NK cells efficiently killed patients' AML blasts at relapse but NK cell activity in patients against their own leukemic blasts was impaired after HSCT. These findings indicate a rationale for donor NK cell infusions after HLA haplo-identical HSCT to avoid decreasing NK cell alloreactivity and to prevent the escape of leukemic cells from allo-immune surveillance by donor cytotoxic T lymphocytes. [Display omitted] Disclosures:No relevant conflicts of interest to declare.