HLA-haploidentical Transplantation Research Articles

Post-Transplantation Cyclophosphamide for Tolerance Induction in HLA-Haploidentical Bone Marrow Transplantation

Allogeneic hematopoietic stem cell transplantation (alloSCT) is a potentially curative therapy for many hematologic and immunologic diseases. Further, partial or full donor hematopoietic chimerism following alloSCT may be sufficient to guarantee immunologic tolerance to solid organs from the same donor, obviating any requirement for prolonged pharmacologic immunosuppression. Despite alloSCT's potential, the procedure is beset by two major limitations. The first relates to the procedure's toxicity, including conditioning regimen toxicity, graft-versus-host disease (GVHD), and infection. The second limitation is the lack of histocompatible donors. A human leukocyte antigen (HLA)-matched sibling or unrelated donor cannot be identified expeditiously for up to 40% of patients. Historically, alloSCT from partially HLA-mismatched, or HLA-haploidentical, relatives has been complicated by unacceptably high incidences of graft rejection, severe GVHD, and non-relapse mortality. Recently, our groups have developed a method to selectively deplete alloreactive cells in vivo by administering high doses of cyclophosphamide in a narrow window after transplantation. Using high-dose, post-transplantation cyclophosphamide (PT/Cy), crossing the HLA barrier in alloSCT is now feasible and donors can be found for nearly all patients. This review discusses the history of HLA-haploidentical SCT, recent clinical results, and immunologic mechanisms of action of high-dose PT/Cy for prevention of graft rejection and GVHD.

Haplo-BMT: cure or back to sickle cell?

In this issue of Blood , Bolanos-Meade et al report promising results using a pioneering HLA-haploidentical bone marrow transplantation protocol in adults with sickle cell disease (SCD), including nonmyeloablative conditioning and post-transplantation high-dose cyclophosphamide. 1

A Novel Regimen of Unmanipulated HLA-Haploidentical Transplantation Using a Small Dose of Anti-T-Lymphocyte Globulin for Patients in High Tumor Burden

AbstractAbstract 4203The major drawback of HLA-haploidentical stem cell transplantation is graft-versus-host disease (GVHD): however, the GVHD can be overcome using substantial T cell depletion method (Perugia), the use of a high dose ATG (Beijing), or the posttransplantation cyclophophamide method (Johns Hopkins). These transplant procedures are found to be effective for the achievement of donor engraftment and immunological tolerance: however, graft-versus-leukemia (GVL) effects are not enough strong to prevent disease relapse. To harness GVL effect of HLA-haploidentical grafts, our regimen for unmanipulated haploidentical myeloablative transplantation is designed for GVH reaction to remain to some extent in the short time after transplantation using a small dose of ATG 2 mg/kg, coupled with GVHD prophylaxis containing a low dose of steroid (methylprednisolone 1 mg/kg). We hypothesize that allogeneic immunological response by donor T cells under low cytokine milieu exerts potent GVL effect without GVHD. This regimen was applied to patients with hematologic disease in an extremely high risk.From August 2008 to June 2012, 23 patients with high-risk or refractory hematologic diseases underwent unmanipulated peripheral blood stem cells using a graft from HLA-haploidentical related donor (11 cases were HLA 2 antigen-mismatched and 12 HLA 3 antigen-mismatched in the GVH direction). Patients’ characteristics are sex: male 11, female 12, age: 17–46 years old (median 34), disease: AML/MDS 7, ALL 10, ML 5, others 1. All patients except for 5 underwent transplantation in non-complete remission (non-CR) status, including 5 patients in induction failure, and 10 in resistant relapse. Eleven patients with leukemia who underwent in non-CR state had a median of 66% blasts in the bone marrow (range, 10.6 – 97.0 %). Patients received a conditioning treatment consisting of fuludarabine, cytarabine, cyclophsphamide, TBI 8Gy and ATG (thymoglobuline) 2 mg/kg. GVHD prophylaxis consisted of tacrolimus and methylprednisolone (1 mg/kg). One patient dies of pneumonia on day 3. Among 22 patients that could be evaluated, the engraftment rate was 95.5% (one patient had graft rejection because of donor-specific HLA antibody). Neutrophil (>0.5×109/l) and platelet (>20×109/l) engraftment was achieved on day 11 and on day 32, respectively. All patients who underwent transplantation in non-CR achieved CR after transplantation. Eleven patients (52.4%) had no GVHD. Nine patients (42.9%) developed grade II-III acute GVHD. One patient died of cardiac failure on day 46, 1 VOD on day 74, and 1 GVHD on day 235. Two patients died of relapse on days 70 and 548. Four of the 8 patients with leukemia who had more than 50% blasts in the bone marrow at the time of transplantation survive in CR. The overall survival is 60% at 3 years (Figure 1).Our regimen for HLA-haploidentical transplantation using a small dose of ATG coupled with low dose of steroid exerts a strong GVL effect even for patients who had chemorefractory disease, and transplantation-related toxicities, including GVHD, are acceptable. As a result, patients, of whom the majority had a high tumor burden, achieved a high rate of long-term survival. Thus, our strategy for haploidentical stem cell transplantation is promising, although our results will have to be confirmed in a large-scale study. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clinical Effect of High-Dose, Donor-Derived Natural Killer Cells Infused After HLA-Haploidentical Hematopoietic Cell Transplantation

AbstractAbstract 1900Haploidentical HCT is feasible without ex vivo T cell depletion after conditioning with reduced-dose busulfan, fludarabine, and ATG (Lee K-H, Blood 2011). Furthermore, infusion of donor-derived NK cells (DNKI) after haploidentical HCT has been shown to suppress leukemia recurrence without GVHD in a murine model. Clinically, DNKI up to 2.5×107/kg have been given to patients after haploidentical HCT without obvious increase in GVHD (Yoon SR, BMT 2010). Clinical effect of higher dose of DNKI, however, remains to be studied. Between February 2009 and February 2012, 37 patients with hematologic malignancies (AML, 28; ALL, 7; MDS, 1; DLBCL, 1) underwent haploidentical HCT and received NK cells derived from the same HCT donors twice at 2 (DNKI#1) and 3 weeks (DNKI#2) after HCT. The donors (median age 29 years, range 7–62; offspring 22, siblings 8, and parents 7) underwent 3 to 4 daily leukapheresis after receiving G-CSF. Donated cells during the initial 2 to 3 days were transplanted without further manipulation. Cells collected on the last day were CD3 depleted and differentiated into NK cells ex vivo. In the initial study phase, as per the protocol design, three-patient cohorts each received 0.2×108/kg, 0.5×108/kg, and 1.0×108/kg donor NK cells twice. Among these initial 9 patients, only one patient who received 1.0×108 cells/kg experienced grade-3 acute GVHD. Thereafter, 3 additional patients received 1.0×108 cells/kg twice and none experienced °Ãgrade-3 acute side effect or acute GVHD. For patients enrolled subsequently, donor NK cell doses were based on the quantity of cells generated. The mean viability of final donor NK cell products for DNKIs #1 and #2 were 85% and 71%, respectively; mean CD56+CD122+, 87% and 94%, respectively; and mean CD3+CD56-, 0.4% and 0.3%, respectively, excluding one case with 16% and 7%. The final cell products exhibited additional NK cell features, such as granzyme/perforin gene expression, NK cell receptor expression, TNF-α/IFN-γ production, and cell cytotoxicity against K562 cells. All 37 patients in the study received at least one DNKI. Four patients did not receive the DNKI#2 (3 due to rapid clinical deterioration and 1 due to cell expansion failure). The median cell doses were 1×108/kg (range, 0.2–2.0 ×108/kg) and 1×108/kg (range, 0.2–4.0 ×108/kg) for DNKIs #1 and #2, respectively. In all patients, DNKI was tolerated well without acute toxicities such as fever or hypotension. After HCT, neutrophil engraftment (cumulative incidence, 87%), grade 2–4 acute GVHD (19%), chronic GVHD (27%), and TRM (29%) were observed. The median follow-up period of survivors was 20.0 months (range, 6.2–41.5). Of 33 patients with refractory leukemia (AML, 25; ALL, 7) or lymphoma (n=1), 7 of 25 with AML and 6 of 8 with ALL/lymphoma experienced progression/recurrence of their underlying malignancies (cumulative incidences, 29% vs. 75%; P=0.0325). When considered together with 31 historical patients with refractory acute leukemia (AML 22; ALL 9) treated with haploidentical HCT without high-dose DNKI, univariate analysis showed that the diagnosis (AML vs ALL/lymphoma, P=0.0048), disease status at HCT (primary refractory vs relapse then refractory, P=0.0410), and patient cohort (study vs historical, P=0.0420) were the significant variable predicting less diseases progression/recurrence. Upon multivariate analysis, the diagnosis (AML vs ALL/lymphoma, P=0.005; odd ratio, 0.356; 95% CI, 0.174–0.729) and patient cohort (study vs historical, P=0.033; odd ratio, 0.471; 95% CI, 0.236–0.941) were independent variables predicting less disease progression/recurrence. Kaplan-Meier event-free and overall survival rates for study AML patients, study ALL/lymphoma patients, historical AML patients, and historical ALL patients were 39% and 36%, 0% and 0%, 7% and 0%, and 0% and 0%, respectively. Our study showed that DNKI with median total dose 2×108/kg given over 2–3 weeks after haploidentical HCT was tolerated well without obvious increase in GVHD or TRM. Furthermore, DNKI after haploidentical HCT may decrease leukemia progression especially in patients with refractory AML. Disclosures:No relevant conflicts of interest to declare.

Lineage–Specific Chimerism and Incidence of Graft-Failure Following T-Cell Replete Haploidentical Transplantation Using Post-Transplant Cyclophosphamide in Eighty-Nine Consecutive Patients From a Single-Center.

Abstract 3030T-cell replete HLA-haploidentical transplantation using post-transplant cyclophosphamide for prevention of GVHD and graft rejection (Haplo-PTCy) has recently emerged as a valid form of alternative-donor transplantation for patients who lack traditional matched-siblings (MSD) or matched-unrelated donors (MUD). We have demonstrated that patients undergoing Haplo-PTCy can have equivalent rates overall and disease-free survival and equivalent or lower cumulative incidences of GVHD and non-relapse mortality to patients transplanted contemporaneously from MSD and MUD at the same center (Bashey et al ASH 2011 abstract #833). In this study we assessed lineage-specific chimerism, together with incidence and outcome of graft-failure in 89 consecutive first Haplo-PTCy performed for hematologic malignancy in our center between Oct 2005 and Jun 2012. Patient characteristics: M 48, F 41; median age 48 (20–74); Diagnosis AML 28, ALL 16, CLL 11, NHL 9, HL 8, CML 8, MDS 7, MPS 2; median number of matched HLA loci were 5/10 (range 5/10 to 8/10); Median CD34+ and CD3+ cell dose infused were 4.01 × 106/kg (0.84–6.27) and 5.35 × 107/kg (1.4–53.82) respectively. Fifty-eight patients received a marrow graft and 31 received G-CSF mobilized PBSC. The preparative regimen was RIC/NST in 59 (fludarabine 30 mg/m2/d d -6 to -2, TBI 200cGy d-1, cyclophosphamide 14.5 mg/kg/d d-6 & -5, and 50 mg/kg/d d+3 & +4) and myeloablative in 30 (regimen A- fludarabine 30 mg/m2/d d -6 to -2, busulfan 110–130 mg/m2/d d-7 to -4, cyclophosphamide 14.5 mg/kg/d d-3 & -2, and 50 mg/kg/g d+3 & +4 [20 patients] and regimen B- fludarabine 30 mg/m2/d d -7 to -5, TBI 1200 cGy given in 8 fractions between days-4 to -1 and cyclophosphamide 50 mg/kg/d d +3 & +4 [10 patients]). The presence of pre-transplant anti-donor HLA antibodies were assessed using a solid phase assay (Panel Reactive Antibody, PRA, Clinimmune, CO) and by anti-donor cross-matching by flow cytometry (Clinimmune). All donors were selected to provide a negative cross-match using recipient serum and donor T-cells prior to transplant. Engraftment was determined using standard CIBMTR criteria. Lineage-specific chimerism was determined using PCR for short tandem repeats on peripheral blood mononuclear cells separated by CD3 and CD33 expression using immunomagnetic beads on d 30,60,90 and 180 following transplant. Median time to ANC > 500/mm3 was 16d (12–27d) and platelets > 20,000/mm3 was 26d (0–26d). Median T-cell (CD3) and myeloid (CD33) donor chimerisms were 100%, at all time-points assessed from d30–180 (Fig 1). All 30 patients who received a myeloablative Haplo-PTCy had full engraftment of T-cells and myeloid cells starting d +30. However six of 59 patients undergoing RIC/NST Haplo-PTCy had primary failure of T-cell engraftment -median CD3 chimerism (range) for these patients on d 30 and 60 were 0% (0–6%) and 0% (0–14%). Median CD33+ cell chimerism for the same patients on d 30 and 60 respectively were 86% (0–100%) and 45% (0–100%). Four of these patients underwent a second Haplo-PTCy, a median of 105d (range 8–123d) following the first transplant using a different haploidentical donor and the same preparative regimen. In each case the second Haplo-PTCy was successful (CD3+ donor chimerism 100% by d 30–60 in all cases). One patient who was too unwell for second Haplo-PTCy had spontaneous improvement in CD3 chimerism (6–14% d 30–90 improving to 100% d 180) and one patient died of progressive malignancy before a second Haplo-PTCy could be performed.These data demonstrate that full donor chimerism of T-cells and myeloid cells is usual following Haplo-PTCy from the earliest time-points assessed. Engraftment failure was not seen in any patient using the myeloablative regimens described above. Approximately 10% of patients conditioned with the RIC/NST regimen failed to undergo initial T-cell engraftment. However, re-transplantation was successful in all cases when attempted. Late spontaneous improvement of CD3 chimerism is also possible in patients with low level mixed chimerism early post-transplant. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Different mechanisms causing loss of mismatched human leukocyte antigens in relapsing t(6;11)(q27;q23) acute myeloid leukemia after haploidentical transplantation

Mismatched human leukocyte antigens (HLAs) on leukemic cells can be targeted by donor T cells in HLA-mismatched/haploidentical stem cell transplantation. In two cases of acute myeloid leukemia with t(6;11)(q27;q23) abnormality presented here, flow cytometry analysis showed a lack of HLA-A unshared between recipients and donors in relapsing leukemic cells after HLA-haploidentical transplantation. However, high-resolution HLA genotyping showed that one case lacked a corresponding HLA haplotype, whereas the other preserved it. These cases suggest that leukemic cells, which lacked mismatched HLA expression, might have an advantage in selective expansion under donor T-cell immune surveillance after HLA-haploidentical transplantation. Most importantly, down-regulation of unshared HLA expression potentially occurs by genetic alterations other than loss of HLA alleles.

HLA-haploidentical bone marrow transplantation with posttransplant cyclophosphamide expands the donor pool for patients with sickle cell disease

Allogeneic marrow transplantation can cure sickle cell disease; however, HLA-matched donors are difficult to find, and the toxicities of myeloablative conditioning are prohibitive for most adults with this disease. We developed a nonmyeloablative bone marrow transplantation platform using related, including HLA-haploidentical, donors for patients with sickle cell disease. The regimen consisted of antithymocyte globulin, fludarabine, cyclophosphamide, and total body irradiation, and graft-versus-host disease prophylaxis with posttransplantation high-dose cyclophosphamide, mycophenolate mofetil, and tacrolimus or sirolimus. After screening 19 patients, we transplanted 17, 14 from HLA-haploidentical and 3 from HLA-matched related donors. Eleven patients engrafted durably. With a median follow-up of 711 days (minimal follow up 224 days), 10 patients are asymptomatic, and 6 patients are off immunosupression. Only 1 patient developed skin-only acute graft-versus-host disease that resolved without any therapy; no mortality was seen. Nonmyeloablative conditioning with posttransplantation high-dose cyclophosphamide expands the donor pool, making marrow transplantation feasible for most patients with sickle cell disease, and is associated with a low risk of complications, even with haploidentical related donors. Graft failure, 43% in haploidentical pairs, remains a major obstacle but may be acceptable in a fraction of patients if the majority can be cured without serious toxicities.

Cyclophosphamide-Based In Vivo T-Cell Depletion for HLA-Haploidentical Transplantation in Fanconi Anemia

Allogeneic hematopoietic cell transplantation (HCT) is the only known cure for patients with Fanconi anemia (FA) who develop aplasia or leukemia. However, transplant regimens typically contain high-dose alkylators, which are poorly tolerated in FA patients. Furthermore, as many patients lack human leukocyte antigen (HLA)-matched family donors, alternative donors are used, which can increase the risk of both graft rejection and graft-versus-host disease (GVHD). To improve on these three concerns, we developed a multi-institutional clinical trial using a fludarabine (FLU)-based conditioning regimen with limited alkylators/low-dose radiation, HLA-haploidentical marrow, followed by reduced-dose cyclophosphamide (CY) to treat three FA patients with aplasia. All three patients engrafted with 100% donor CD3 chimerism at 1 month. One patient died early from disseminated toxoplasmosis infection. Of the two survivors, one had significant pretransplant co-morbidities and inadequate immunosuppression, and developed severe acute GVHD. The other patient had only mild acute and no chronic GVHD. With a follow-up of 2 and 3 years, respectively, both patients are doing well, are transfusion-independent, and maintain full donor chimerism. The patient with severe GVHD has resolving oral GVHD and good quality of life. We conclude that using low-intensity conditioning, HLA-haploidentical marrow, and reduced-dose CY for in vivo T-cell depletion can correct life-threatening aplasia in FA patients.

HLA-haploidentical transplantations for primary immunodeficiencies: A single-center experience

SCID is characterized by profound deficiencies of T and B lymphocytes. HSCT is the only curative treatment for children with SCID. The clinical characteristics and outcome of 30 HLA-haploidentical transplantations in 18 patients (15 SCID, two Omenn syndrome, and one MHC Class II deficiency) are reported here. The age of patients at diagnosis ranged from one and half to nine months (median: four months). The median time was one month between the diagnosis and the time of the initial transplantation. Infused CD34+ stem cell dose was ranged between 7 and 94.2 × 10(6) /kg. Nine of 18 patients were found to be positive for CMV antigenemia at diagnosis; therefore, none of them received a conditioning regimen. The most common complication was graft failure (61%), so repeated transplantations (two to four) were performed in seven patients. The mean time of lymphoid engraftment was 17.5 days (median: 16, range: 11-29 days). Ten of 15 SCID (67%) patients survived with a stable complete donor chimerism. However, all three non-SCID patients died. In conclusion, in the absence of a matched family donor, HLA-haploidentical transplantation from parental donors represents a readily available treatment option especially for patients with SCID, offering a high chance of cure.

Indirect evidence that maternal microchimerism in cord blood mediates a graft-versus-leukemia effect in cord blood transplantation

During pregnancy women can develop B- and T-cell immunity against the inherited paternal antigens (IPAs) of the fetus, such as HLA, peptides of minor histocompatibilty antigens, and possibly onco-fetal antigens. The biological and pathological role of these pregnancy-induced immunological events is only understood in part. However, anti-IPA immunity in the mother persists for many decades after delivery and may reduce relapse in offspring with leukemia after HLA-haploidentical transplantation of maternal hematopoietic stem cells (HSC). We hypothesized that maternal anti-IPA immune elements cross the placenta and might confer a potent graft-versus-leukemia effect when cord blood (CB) is used in unrelated HSC transplantation. In a retrospective study of single-unit CB recipients with all grafts provided by the New York Blood Center, we show that patients with acute myeloid or lymphoblastic leukemia (n = 845) who shared one or more HLA-A, -B, or -DRB1 antigens with their CB donor's IPAs had a significant decrease in leukemic relapse posttransplantation [hazard ratio (HR) = 0.38, P < 0.001] compared with those that did not. Remarkably, relapse reduction in patients receiving CB with one HLA mismatch (HR = 0.15, P < 0.001) was not associated with an increased risk of severe acute graft-versus-host disease (HR = 1.43, P = 0.730). Our findings may explain the unexpected low relapse rate after CB transplantation, open new avenues in the study of leukemic relapse after HSC transplantation (possibly of malignancies in general), and have practical implications for CB unit selection.

Unmanipulated Donor Lymphocytes for EBV-Related PTLD After T-Cell Depleted HLA-Haploidentical Transplantation

Epstein-Barr virus (EBV)-related post-transplantation lymphoproliferative disorder (PTLD) is a life-threatening complication in patients given T-cell-depleted hematopoietic stem cell transplantation from an HLA-haploidentical relative (haplo-HSCT). We report the case of a child who developed severe EBV-related PTLD after haplo-HSCT from his mother. Despite receiving the anti-CD20 monoclonal antibody, the patient presented with intestinal obstruction due to huge abdominal lymphadenopathy, hematemesis, and nodulary pulmonary lesions. Histology showed that the lesions were due to CD20-/CD19+ large neoplastic B cells. The patient underwent double intestinal resection with partial abdominal lymphadenectomy and then received 3 monthly doses of donor-derived unmanipulated mononuclear cells. The initial dose of CD3+ cells was 310(5)/kg recipient body weight. The 2 additional doses consisted of 510(5) CD3+ cells/kg. No sign or symptom attributable to graft-versus-host disease was observed, and the patient completely cleared EBV-related lesions. The child was disease-free for 13 months after the first lymphocyte infusion. This case demonstrates that repeated infusions of controlled numbers of donor CD3+ cells cure EBV-related PTLD in haplo-HSCT without inducing graft-versus-host disease.

Favorable Immune Reconstitution After Nonmyeloablative, T-Cell Replete, HLA-Haploidentical BMT with Post-Transplant Cyclophosphamide

Abstract 1009Delayed immune reconstitution with increased risk of opportunistic infection is a major complication of HLA-haploidentical stem cell transplantation, especially in protocols employing extensive T cell depletion of the graft. Previous studies at our institution with high-dose, post-transplantation Cy (PT/Cy) have reported low rates of non-relapse mortality and serious opportunistic infections. Here we characterize immune reconstitution in fifty-three consecutive hematologic malignancies patients receiving nonmyeloablative conditioning, T cell-replete, HLA-haploidentical bone marrow transplantation (BMT), and graft versus host disease prophylaxis including PT/Cy. Patients with advanced hematologic malignancies (median age 51, range 14–71; 5 AML, 2 ALL, 4 MDS, 2 CML, 4 CLL, 1 CMML, 25 NHL, 7 Hodgkins, 3 mantle cell) received Cy 14.5 mg/kg/day IV on days −6 and −5, fludarabine 30 mg/m2/day IV on days −6 to −2, 200 cGy of TBI on day -1 and T cell replete bone marrow from donors with a median age of 44 (range 14–68). GVHD prophylaxis consisted of Cy (50 mg/kg/day) on days 3 and 4, mycophenolate mofetil for 30 days, and tacrolimus for 6 months. Grafts contained an infused median TNC/kg of 4.1 e8 (range 2.6–6.6 e8), CD3+/kg 3.6 e7 (range 1.7–6.7e7) and a CD34+/kg of 3.5e6 (range 1.4–7.0e6). Sustained engraftment of donor cells occurred in 86% of evaluable patients (44/51).The median times to neutrophil (>500/μL) and platelet recovery (>20,000/μL) were 17 days (range, 13–92 days) and 28 days (range, 13–580 days), respectively. Post-transplantation recovery of lymphocyte subsets is shown in Table 1 and Figure 1 and is notable for the following: 1) The median lymphocyte count at day 30 after transplantation is >180/ml and recovers to over 800/ml by day 60; 2) CD4+ T cell counts recover to a median >120/ml by day 60 and >220/ml by day 180 after transplantation; and 3) recovery of CD31+ recent thymic emigrants and CD45RA+ naïve T cells is delayed compared to recovery of memory T cells. T cell receptor spectratyping analysis on a subset of 10 patient/donor pairs chosen specifically for having no relapse/no GVHD (n=4), GVHD and no relapse (n=3), or late relapse (n=3) revealed that patients without relapse, GVHD, or recent viral infection had excellent reconstitution of the T cell repertoire to the level of the pre-transplant donor, as early as 6 months post-transplant (Figure 2). CMV specific T cell response using ELISPOT measured on a subset of 17 patients whose donors were reactive to CMV, revealed that donor-derived immunity to CMV returns by Day 60 in about 70% of patients (12/17) (Figure 3). In conclusion, immune reconstitution after non-myeloablative haploidentical T cell replete BMT with PT/Cy compares favorably with other reduced intensity conditioning alternative donor regimens and suggests that PT/Cy selectively preserves pathogen-specific memory T cells necessary to protect against infection. Further correlations of immune reconstitution with specific infectious and overall outcomes are being analyzed. [Display omitted] [Display omitted] [Display omitted] Table 1Post-transplantation Lymphocyte Subset RecoveryMedian (cells/μL) (N)Interquartile range (cells/μL)ALCDonor1765 (46)1480–2100Recipient pre-BMT840 (45)425–1295Day 30184 (49)54–402Day 60820 (38)470–1260Day 180915 (34)670–1560Day 3653060 (22)820–2030CD3+CD4+CD45RA+ (naïve)Donor119 (33)82–189Recipient pre-BMT22 (34)4–38Day 300.33 (35)0.07–1Day 603 (29)1–9Day 18011 (23)5–31Day 36523 (13)13–92CD3+CD4+CD45RA−CCR7+ (central memory)Donor135 (33)95–158Recipient pre-BMT54 (34)11–79Day 302 (35)0.5–11Day 6034 (29)10–79Day 18061 (23)35–117Day 36589 (13)60–122CD3+CD4+CD45RA−CCR7− (effector memory)Donor187 (33)130–245Recipient pre-BMT87 (34)14–134Day 303 (35)1–18Day 6059 (29)14–122Day 180102 (23)43–179Day 365142 (12)64–204CD3+CD4+CD45RA+CD31+ (recent thymic emigrants)Donor61 (33)30–97Recipient pre-BMT6 (34)1–16Day 300.9 (35)0.03–0.4Day 601 (29)0.5–2Day 1804 (23)1–11Day 3657 (13)2–12CD3+CD4+Foxp3+Donor28 (33)23–35Recipient pre-BMT13 (34)6–24Day 301 (35)0.1–5Day 608 (29)4–16Day 18013 (23)7–25Day 36514 (13)7–17ALC, absolute lymphocyte count; WBC, white blood cell count; Treg, regulatory T cell Disclosures:Jones:Aldagen: Patents & Royalties.

Adoptive Immunotherapy with Tregs and Tcons Ensures Low TRM and a Low Incidence of Post Transplant Leukaemia Relapse After HLA Haploidentical Transplants for Acute Leukemia

Abstract 154In full haplotype mismatched (HLA-haploidentical) stem cell transplantation we showed adoptive transfer of freshly isolated donor CD4+CD25+ FoxP3+ T regulatory cells (Tregs) followed by donor T cells (Tcons) prevented acute and chronic GvHD without any post-transplant immunosuppression, promoted lymphoid reconstitution and improved immunity against opportunistic pathogens (Di Ianni et al., Blood 2011). The major drawback was the extra-haematological toxicity of the conditioning regimen which included TBI, thiotepa, fludarabine and cyclophosphamide. To reduce regimen related toxicity we replaced cyclophosphamide with alemtuzumab, given 22 days before the Treg infusion to prevent it from interfering with adoptive T cell immunotherapy (Fig 1). The graft consisted of immunoselected Tregs (median 2×106/kg; range 1.6–4.8; FoxP3+ cells 92% ± 8 SD;), CD34+ cells (median 9.1×106/kg; range 8.1–10.9) and Tcons (median 1×106/kg; range 0.5–3). No post-transplant prophylaxis against GvHD was given. [Display omitted] Since May 2010 18 patients (median age 43 years, range 23–61) with high risk acute leukaemia (16 AML, 2 ALL) have been transplanted. All sustained full donor-type engraftment. Neutrophils reached 0.5×109/L at a median of 12 days (range 9–28 days). Platelets reached 20×109/L and 50×109/L at median of 12 and 15 days, respectively (range 10–36 days and 11–55 days). CD4+ and CD8+ peripheral blood counts reached, respectively, 50/μL medianly on days 36 (range 27 – 120 days) and 34 (range 15– 85); 100/μL medianly on days 55 (range 27 – 147 days) and 48 (range 27 – 114); 200/μL on days 62 (range 37 – 177 days) and 49 (range 28 – 147). We observed a rapid development of a wide T-cell repertoire with specific CD4+ and CD8+ T cells for opportunistic pathogen antigen such as Aspergillus, Candida, CMV, ADV, HSV, VZV, Toxoplasma. Treg immunotherapy did not compromise post-transplant generation of donor-vs-recipient alloreactive natural killer (NK) cell repertoires in patients who received transplants from NK alloreactive donors (Ruggeri et al., Science 2002).Three of 16 valuable patients developed acute GvHD. Two responded to a short course of immunosuppressive therapy and at present (288 and 360 days after transplant) are alive and well with very good immunological reconstitution. The 3rd patient died of infectious complications. Two other patients died of non-leukemic causes (1 fulminant hepatitis 17 days post-transplant, 1 pneumonia 14 days post-transplant). The incidence of TRM is 17% (3/18). As hoped, extra-haematological toxicity was mild. One AML patient, who received a transplant from a non-NK alloreactive donor, relapsed 77 days post-transplant. Fourteen of the 18 patients are alive and well at a minimum follow-up of 3 months.This study shows adoptive immunotherapy with freshly isolated, naturally occurring Tregs is a feasible option in HLA-haploidentical stem cell transplantation since alloantigen-specific Tregs were efficiently activated in vivo and controlled alloreactivity of at least 1×106/kg Tcons without clinically significant inhibition of general immunity.Moreover Treg infusion did not weaken the GvL effect. The incidence of post-transplant leukaemia relapse was surprisingly low as only 1 patient has relapsed to date and even in our previous series no patient who was transplanted in CR has relapsed at a median follow-up of 25 months. Infusion of high numbers of Tcons in the absence of post-transplant immunosuppression can be hypothesized to exert a GvL effect. In addition, in patients who were transplanted from NK alloreactive donors, preservation of alloreactive NK cell repertoires played a key role in reducing the incidence of relapse. Disclosures:No relevant conflicts of interest to declare.

Tregs combined with mature donor T cells hasten immune reconstitution without triggering GvHD in HLA haploidentical transplantation

Haploidentical transplantation, with extensive T cell depletion to prevent GvHD, is associated with a high incidence of infection-related deaths. The key challenge is to improve immune recovery with allogeneic donor T cells without triggering GvHD. As T regulatory cells (Tregs) controlled GvHD in preclinical studies, the present phase I/II clinical trial evaluated the impact of early infusion of donor CD4/CD25+ Tregs, followed by an inoculum of donor mature T cells (Tcons) and positively immunoselected CD34+ cells. Twenty-eight patients (median age 41, range 21-60) were enrolled from September 2008 onwards; 22 had AML (10 in CR1 at high risk, 10 in ≥CR2 and 2 in relapse), 5 had ALL (4 in CR1; 1 in relapse) and 1 had high grade NHL in relapse. Conditioning was: 8 Gy single fraction TBI, thiotepa (4 mg/kg × 2), fludarabine (40 mg/m2 × 5), cyclophosphamide (35 mg/kg × 2). All patients received immunoselected Tregs (CliniMACS, Miltenyi Biotec) (23/28 2 × 106/kg bw; 5/28 4 × 106/kg bw) and 4 days later positively immunoselected CD34+ cells (median 8.2 × 106/kg bw, range 5.0-19.1) together with Tcons (4/28 0.5 × 106/kg bw; 17/28 1 × 106/kg bw; 5/28 2 × 106/kg bw; 2/28 did not receive Tcons). CD4/CD25+ Tregs (purity 92.7 ± 2.1) consisted of 33.6% ± 13.1 CD25high; 58.1% ± 6.6 CD25int; 5.8% ± 2.5 CD25low; 65.7% ± 11.8 FoxP3; 17.4% ± 7.2 CD127 (mean ± SD). No GvHD prophylaxis was administered. 26/28 patients engrafted. No GvHD developed in 24/26 patients, 2 developed ≥ grade II GvHD. Ten patients died (3 VOD, 2 fungal pneumonia, 1 bacterial sepsis, 1 CNS aspergillosis, 1 systemic toxoplasmosis, 1 adenoviral infection, 1 MOF). CD4 and CD8 counts reached, respectively, 50/μL medianly on days 34 (range 19-63 days) and 24 (range 15-87); 100/μL medianly on days 47 (range 28-100 days) and 34 (range 19-95); 200/μL on days 70 (range 41-146 days) and 61 (range 21-95). A wide T-cell repertoire developed rapidly with high frequencies of specific CD4+ and CD8+ for opportunistic pathogens. Episodes of CMV reactivation were significantly fewer than after our standard haplo transplants. In KIR ligand-mismatched transplants, speed of NK cell reconstitution/maturation and size of donor vs recipient alloreactive NK cell repertoires were preserved. In conclusion, in the setting of haploidentical transplantation infusion of Tregs makes administration of a high dose of T cells feasible for the first time. This strategy provides a long-term protection from GvHD and robust immune reconstitution.

The effect of HLA disparity on clinical outcome after HLA-haploidentical blood and marrow transplantation

The relative importance of various human leukocyte antigen (HLA) loci has not been established for unmanipulated HLA-mismatched/haploidentical transplantation. To address this question, we analyzed the impact of HLA-A, HLA-B, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 on the outcome of HLA-haploidentical transplantation. Four hundred and eighty-one donor-recipient pairs were fully typed before transplantation. In univariate analysis, HLA-B mismatch not only demonstrated significant adverse effects on acute graft-versus-host disease (GVHD) and transplant-related mortality but also was associated with reduced overall survival and leukemia-free survival (LFS). In multivariate analysis, HLA-B mismatch remained the independent risk factor for acute GVHD and transplant-related mortality. The high risk of disease and the female donor were found to be significant factors for reduced overall survival and LFS. Furthermore, multiple mismatch of the HLA locus was found to have no synergistic adverse effect on outcomes. Our results suggest that prospective matching of patients and donors for HLA-B antigen in the unshared HLA haplotype is warranted for HLA-mismatched/haploidentical transplantation.

Allogeneic hematopoietic cell transplantation in patients with myelodysplastic syndrome and concurrent lymphoid malignancy

Allogeneic hematopoietic cell transplantation (HCT) can be curative for both myelodysplastic syndromes (MDS) and lymphoid malignancies. Little is known about the efficacy of allogeneic HCT in patients in whom both myeloid and lymphoid disorders are present at the time of HCT. We analyzed outcomes in 21 patients with MDS and concurrent lymphoid malignancy when undergoing allogeneic HCT. Seventeen patients had received extensive prior cytotoxic chemotherapy, including autologous HCT in seven, for non-Hodgkin lymphoma (NHL, n=7), Hodgkin lymphoma (HL, n=2), chronic lymphocytic leukemia (CLL, n=5), NHL plus HL (n=1), multiple myeloma (n=1), or T-cell acute lymphocytic leukemia (ALL) (n=1), and had, presumably, developed MDS as a consequence of therapy. Four previously untreated patients had CLL. Nineteen patients were conditioned with high-dose (n=14) or reduced-intensity regimens (n=5), and transplanted from HLA-matched or one antigen/allele mismatched related (n=10) or unrelated (n=9) donors; two patients received HLA-haploidentical related transplants following conditioning with a modified conditioning regimen. Currently, 2 of 4 previously untreated, and 2 of 17 previously treated patients are surviving in remission of both MDS and lymphoid malignancies. However, the high non-relapse mortality among previously treated patients, even with reduced-intensity conditioning regimens, indicates that new transplant strategies need to be developed.

Reduced-intensity conditioning therapy with busulfan, fludarabine, and antithymocyte globulin for HLA-haploidentical hematopoietic cell transplantation in acute leukemia and myelodysplastic syndrome

AbstractAny role for reduced-intensity conditioning (RIC) before hematopoietic cell transplantation (HCT) from a human leukocyte antigen (HLA)–haploidentical donor remains to be defined. We therefore assessed 83 patients (age, 16-70 years): 68 with acute leukemia (including 34 in remission and 34 with refractory disease) and 15 patients with myelodysplastic syndrome, in HCT trials using RIC with busulfan, fludarabine, and antithymocyte globulin. The HLA-haploidentical donors, offspring (n = 38), mothers (n = 24), or siblings (n = 21) of patients, underwent leukapheresis after receiving granulocyte colony-stimulating factor, and donated cells were transplanted without further manipulation. Cyclosporine and methotrexate were given for GVHD prophylaxis. The cumulative incidences of neutrophil engraftment, grade 2 to 4 acute GVHD, chronic GVHD, and transplantation-related mortality after HCT, were 92%, 20%, 34%, and 18%, respectively. After a median follow-up time of 26.6 months (range, 16.8-78.8 months), the event-free and overall survival rates were 56% and 45%, respectively, for patients with acute leukemia in remission; 9% and 9%, respectively, for patients with refractory acute leukemia; and 53% and 53%, respectively, for patients with myelodysplastic syndrome. HCT from an HLA-haploidentical family member resulted in favorable outcomes when RIC containing antithymocyte globulin was performed. This study is registered at www.clinicaltrials.gov as #NCT00521430 and #NCT00732316.

Nonmyeloablative, HLA-Haploidentical Bone Marrow Transplantation with High Dose, Post-Transplantation Cyclophosphamide

Allogeneic stem cell transplantation (SCT) from an HLA-haploidentical relative provides a potentially curative treatment option for hematologic malignancies patients who lack a suitably HLA-matched donor. The greatest challenge to performing HLA-haploidentical SCT has been high rates of graft failure and severe graft-versus-host disease (GVHD). Our group has been exploring high dose, post-transplantation cyclophosphamide (Cy) as prophylaxis of GVHD after nonmyeloablative, HLA-haploidentical bone marrow transplantation, or mini-haploBMT. Among 210 recipients of mini-haploBMT, 87% of patients have experienced sustained donor cell engraftment. The cumulative incidences of grades II-IV acute GVHD and chronic GVHD are 27% and 13%, respectively. Five-year cumulative incidence of non-relapse mortality is 18%, relapse is 55%, and actuarial overall survival and event-free survivals are 35% and 27%, respectively. These outcomes suggest that mini-haploBMT with post-transplantation Cy is associated with acceptably low toxicities and can provide longterm survival, if not cure, for many patients with advanced hematologic malignancies.

Salvage haploidentical transplantation for graft failure using reduced-intensity conditioning

Graft failure is a major concern after cord blood transplantation (CBT) or HLA-haploidentical transplantation (haplo-SCT). As patients who undergo CBT or haplo-SCT almost always lack both matched-related and -unrelated donors, salvage transplantation would also be limited to either CBT or haplo-SCT. In this study, we assessed eight patients who received haplo-SCT as salvage therapy for graft failure. Five and three patients had received haplo-SCT and CBT, respectively, which resulted in graft failure. The median interval from the failed transplantation to salvage transplantation in six patients with primary graft failure was 33.5 days. The reduced-intensity conditioning regimen consisted of fludarabine, thiotepa, rabbit antithymocyte globulin and low-dose TBI. All eight patients achieved neutrophil engraftment, and seven patients achieved platelet recovery. The median times to neutrophil recovery and platelet recovery were 10 and 20 days, respectively. Three patients died from treatment-related causes: two from GVHD and one from rupture of carotid artery aneurysm. Five patients are alive, at a median follow-up of 946 days. The probability of overall survival at 5 years was 75%. These findings may serve as a rationale for giving precedence to haplo-SCT over CBT in salvage SCT after graft failure.

Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation

AbstractHastening posttransplantation immune reconstitution is a key challenge in human leukocyte antigen (HLA)–haploidentical hematopoietic stem-cell transplantation (HSCT). In experimental models of mismatched HSCT, T-regulatory cells (Tregs) when coinfused with conventional T cells (Tcons) favored posttransplantation immune reconstitution and prevented lethal graft-versus-host disease (GVHD). In the present study, we evaluated the impact of early infusion of Tregs, followed by Tcons, on GVHD prevention and immunologic reconstitution in 28 patients with high-risk hematologic malignancies who underwent HLA-haploidentical HSCT. We show for the first time in humans that adoptive transfer of Tregs prevented GVHD in the absence of any posttransplantation immunosuppression, promoted lymphoid reconstitution, improved immunity to opportunistic pathogens, and did not weaken the graft-versus-leukemia effect. This study provides evidence that Tregs are a conserved mechanism in humans.