Stable Engraftment Research Articles

Successful Retransplantation of Bone Marrow Cells Following Failure of Initial Engraftment in 4 Patients with SCN

Severe congenital neutropenia (SCN) is characterized by severe chronic neutropenia with a absolute neutrophil count of less than 0.5×109/L, maturation arrest of myeloid precursors at the promyelocyts/myelocyte stage, and the recurrent bacterial infections from early infancy. The induction of G-CSF therapy dramatically improved the prognosis of most patients with SCN. Analysis on patients on the Severe Chronic Neutropenia International Registry has demonstrated that the patients with SCN on long-term G-CSF treatment are at a risk of developing myelodysplastic syndrome or acute myeloid leukemia (MDS/AML). Recently, hematopoietic stem cell transplantation (HSCT) has been applied for a curative treatment in SCN patients without malignant transformation. However, the optimal conditions of HSCT for SCN have not been established. The failure of engraftment has been reported in some of SCN patients receiving HSCT with reduced intensity conditioning (RIC) or using cord blood as a donor source. Here, we tried to undergo retransplantation of bone marrow cells for 4 SCN patients with ELANE mutation who referred to our hospital because of the engraftment failure of initial HSCT. The summary of initial HSCT is shown in Table. The source of stem cells and the conditioning regimen were heterogenous in four patients. In this study we used bone marrow cells as stem cell source for all patients to transfuse the sufficiently total nucleated cells. Bone marrow cells were obtained from an HLA-matched related, an HLA-matched unrelated, and two HLA-mismatched unrelated (7/8) donors, respectively. Conditioning regimen consisted of fludarabine (125 mg/m2), cyclophosphamide (140 mg/kg), anti-thymocyte globulin (ATG, 10-12 mg/kg), melpharan (90 mg/m2), and TLI (3.6 Gy). Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft versus host disease (GVHD). Engraftment of neutrophils was observed within post-transplant 21 days in all patients. Three of 4 patients have achieved complete engraftment without any signs of GVHD during their course. One patient required additional donor-lymphocyte infusion to sustain the stable engraftment. All patients are alive for 1 to 5 years after HSCT with no signs of infections or transplantation-related morbidity. Furthermore, we observed successful BMT using similar conditioning regimen in other 4 SCN patients without prior transplantation. In our cohort, sufficient administration of ATG is thought to be critical for preventing both engraftment failure and acute GVHD. These results suggest that bone marrow transplantation using fludarabine-based conditioning regimen with a sufficient dose of ATG may be a feasible and effective treatment for SCN patients irrespective of initial engraftment failure. Indications for proceeding to HSCT have been under discussion in patients with SCN. Accumulation of cases and further analysis are necessary to assess the efficacy and safety of this regimen including late complication related to HSCT.TableHSCT characterictecs at the initial transplantationCase1234EtiologyELANE G185RELANE V72MELANE 4501-4503 delELANE V72MAge at the time of HSCT10 months2 years3 years 9 months15 monthsStem cell sourcecord bloodbone marrowcord bloodbone marrowHLA disparitymismatched unrelated donor (5/6)matched unrelated donor (6/6)matched related donor (6/6)mismatched unrelated donor (6/8)Transfused NCC (108/kg)0.71.90.491Transfused CD34 cells (106/kg)0.6991.90.071.07Conditioning regimenBU 16, CY 120, Flu 120CY 140, Flu 125, L-PAM 90 TBI 3, ATG 2.5CY 100, Flu 120 TBI 3, ALG 40Flu 125, L-PAM 140 TBI 2, ATG 5Posttransplant day of rejection72 days55 days after 4 times of DLI14 daysone year after mixed chimerismDLI (times)0430Busulfan (BU, mg/kg), Cyclophophamide (CY, mg/kg), Fludarabine (Flu, mg/m2), Melpharan (L-PAM, mg/m2), Total body irradiation(TBI, Gy), Anti-thymoglobulin (ATG, mg/kg), Anti-lymphoglobulin (ALG, mg/kg), donor lymphocyte infusion (DLI) DisclosuresNo relevant conflicts of interest to declare.

Abstract 1001: CD146 is a novel marker of highly tumorigenic populations and a therapeutic target in malignant rhabdoid tumor

Abstract Background: Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy, particularly develops during infancy and early childhood. In contrast to significant advances in multimodality therapy for other pediatric malignancies, effective therapies for MRT have not been established. The origin of tumor cells in MRT has remained undetermined: however, MRT arises from various parts of body with some preference, eading to the hypothesis that MRT derives from neural crest or its derivatives. CD146 is a cell surface glycoprotein expressed by various neural crest-derived tissues, which has been expected to be a therapeutic target for immunotherapy against various CD146-expressing malignancies, such as melanoma and osteosarcoma. Objective: In the current study, we investigated (1) the expression of CD146 in MRT, (2) tumorigenic potential of CD146-expressing cells in MRT, (3) in vitro and in vivo effect of anti-CD146 neutralizing antibody against MRT, and (4) underlying molecular mechanisms involved in CD146 expression. Material and Method: The expression levels of CD146 in MRT cell lines and clinical samples were investigated by fluorescence-activated cell sorting (FACS) or immunohistochemical analyses. Tumor cells were then sorted by FACS according to the expression of CD146, and purified CD146+ and CD146- cells were characterized by colony-formation, sphere-formation, and invasion assays. In vivo tumorigenic potential was assessed by subcutaneous transplantation of the purified CD146+ and CD146- cells into NOD/Shi-scid, IL-2Rγnull (NOG) mice. We developed rabbit anti-CD146 neutralizing antibody to investigate the effect of blocking CD146 both in vitro and in vivo. DNA microarray was performed by using purified CD146+ and CD146- cells, and the association of candidate pathways was validated by shRNA silencing or treatment of the neutralizing antibody against CD146. Results: The expression of CD146 was observed in all MRT cell lines and clinical samples. Compared to CD146- cells, CD146+ cells in MRT exhibited significantly higher colony-, and sphere-forming potential, and more invasive capacity. The data of xeno-transplant demonstrated that stable engraftment of the purified tumor cells as well as successful engraftment after serial transplantation was exclusively observed in CD146+ cell-injected mice. Furthermore, our anti-CD146 neutralized antibody effectively inhibited both in vitro proliferation and in vivo tumor formation. Microarray analysis showed that various molecular pathways were engaged in higher tumorigeneity in CD146+ cells. Details of underlying mechanisms are now under investigation. Conclusion: Our data shows that CD146 defines a distinct subpopulation with highly tumorigenic and self-renewal capacity in MRT. Notable anti-tumor effect of anti-CD146 neutralizing antibody will facilitate a novel immunotherapy for MRT. Citation Format: Seishiro Nodomi, Katsutsugu Umeda, Satoshi Saida, Yasumichi Kuwahara, Takayuki Hamabata, Tomoo Daifu, Itaru Kato, Hidefumi Hiramatsu, Ken-ichiro Watanabe, Souichi Adachi, Eiichi Konishi, Hajime Hosoi, Toshio Heike. CD146 is a novel marker of highly tumorigenic populations and a therapeutic target in malignant rhabdoid tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1001. doi:10.1158/1538-7445.AM2014-1001

Stable long-term mixed chimerism achieved in a canine model of allogeneic in utero hematopoietic cell transplantation

AbstractEvidence supporting the efficacy of in utero hematopoietic cell transplantation (IUHCT) in a valid large animal model is needed prior to clinical application. The objective of this study was to establish clinically relevant levels of hematopoietic chimerism in a canine model of maternal-to-fetal IUHCT. We first assessed immune and hematopoietic ontogeny relevant to IUHCT in the canine model and identified 40 days’ gestation (term 63 days) as a time point at the initiation of thymic selection, and prior to bone marrow hematopoiesis, that might be optimal for IUHCT. We next determined that intravascular administration of donor cells via intracardiac injection was far more efficient and resulted in much higher levels of donor cell engraftment than intraperitoneal injection. By applying these findings, we achieved stable long-term multilineage engraftment in 21 of 24 surviving recipients with an average level of initial chimerism of 11.7% (range 3% to 39%) without conditioning or evidence of graft-versus-host disease. Donor cell chimerism remained stable for up to 2 years and was associated with donor-specific tolerance for renal transplantation. The levels of donor cell chimerism achieved in this study would be therapeutic for many hematopoietic disorders and are supportive of a clinical trial of IUHCT.

Unrelated CD3/CD19-Depleted Peripheral Stem Cell Transplantation for Hurler Syndrome

For patients with mucopolysaccharidosis type IH (MPS1-H; Hurler syndrome), early allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice. One boy and one girl aged 20.5 and 22 months, respectively, with MPS1-H received a conditioning regimen consisting of thiotepa, fludarabine, treosulfan, and ATG. Grafts were peripheral blood stem cells from unrelated donors (10/12 and 11/11 matched), that were manipulated by CD3/CD19 depletion and contained 20.3 and 28.2 × 106 CD34+ cells/kg body weight, respectively. Both patients achieved stable hematopoietic engraftment and stable donor chimerism. Neither acute or chronic graft-versus-host disease (GVHD) nor other severe transplant-related complications occurred. At a follow-up of 48 and 37 months, both patients are alive and well with normal levels of α-L-iduronidase and have made major neurodevelopmental progress. Treosulfan-based conditioning offers the advantage of reduced toxicity; the use of unrelated CD3/CD19-depleted peripheral stem cell grafts allows transfusion of high CD34+ cell numbers together with a “tailored” number of CD3+ cells as well as engraftment facilitating cells in order to achieve rapid hematopoietic engraftment while reducing the risk of graft rejection and GVHD. This regimen might be an additional option when unrelated donor HSCT is considered for a patient with MPS1-H.

Clinical study of unrelated umbilical cord blood transplantation for 25 patients with hematological diseases

This study was purposed to investigate the engraftment, graft-versus-host disease (GVHD), transplantation related mortality (TRM), relapse and survival in hematologic patients received unrelated umbilical cord blood transplantation (UCBT). A total of 25 patients with hematological disease underwent UCBT, including 8 pediatric and 17 young adult patients. Among them 3 cases received single unit of UCBT and 22 cases received double units of UCBT. For donor/recipients human leukocyte antigen (HLA) matching: HLA 6/6 loci matched in 9 cases, HLA 4-5/6 loci matched in 16 cases. There were 19 patients with hematologic malignancies, including 3 cases in the period of disease progression and 6 cases of non-hematologic malignancies. Conditioning regimens were TBI/Cy ± Flu ± ATG or BuCy ± Flu ± ATG for 21 patients and Cy+Flu+ATG for 4 patients. For prophylaxis of acute graft-versus-host disease (aGVHD) the regimen of cyclosporine (CsA) as dominant drug was used. The results showed that among 16 patients (80.0%) achieved engraftment, 20 patients survived for more than 42 d after transplantation. The cumulative neutrophil recovery rate on day 42 after transplant was 64.0%, with a median time of 17.0 d;the cumulative platelet recovery rate on day 100 after transplant was 60.0 %, with a median time of 35.0 d. The cumulative rate of grade II-IV and III-IV aGVHD after transplantation 100 d was 44.0% and 30.7%, respectively. Until the end of the follow-up, the cumulative rate of TRM was 54.3%. For all the patients, overall survival rate was 42.7%. Out of 17 evaluable patients with hematologic malignancies 7 cases (41.2%) survived to date, and only 1 case relapsed, so event-free survival rate was 35.3%. Out of 5 evaluable patients with non-hematologic malignancies, 4 patients survived and 2 patients were in stable engraftment state, 2 cases with autologous hematopoietic recovery. Among 3 cases of hematologic malignancies at advanced stage, only 1 case survived to date. It is concluded that HLA-4-6/6 loci matched UCBT is an effective option to treat hematological diseases. Double cord blood transplantation (dUCBT) can overcome the disadvantage of insufficient cells of single cord blood UCBT to treat overweight children and adult.

In Vivo Environment Necessary to Support Transplanted Donor Mouse T Regulatory Cells

CD4(+) Foxp3(+) T regulatory cells (Tregs ) are essential for maintaining immunological tolerance, which could be harnessed for novel cell-based therapies to prevent allograft rejection and control autoimmunity. However, the use of Tregs for therapy is hindered by the inability to generate sufficient cell numbers to inhibit desired immune response(s) and achieve stable engraftment of the donor-Treg cell inoculums. The present study was undertaken to investigate the in vivo requirements to promote engraftment of adoptively transferred Tregs and induce tolerance. We established that not only is peripheral space required, but competition from endogenous Tregs must be minimized for successful donor-Treg engraftment with IL-2 critical for driving their proliferation and survival. Moreover, these studies revealed a critical level of donor-Treg engraftment was required for tolerance induction to skin transplants. These mouse studies lay the foundation for development of novel Treg approaches for tolerance induction in the clinic involving not only organ or cellular transplantation, but also to re-establish self-tolerance in autoimmune settings.

Total Body Irradiation and Cyclophosphamide Plus Antithymocyte Globulin Regimen Is Well Tolerated and Promotes Stable Engraftment as a Preparative Regimen before T Cell–Replete Haploidentical Transplantation for Acute Leukemia

We compared total body irradiation (TBI, 700 cGy)/cyclophosphamide (Cy, 3.6 g/m2)/simustine (250 mg/m2) plus antithymocyte globulin (ATG) (TBI/Cy plus ATG) with cytarabine (8 g/m2)/i.v. busulfan (Bu, 9.6 mg/kg)/Cy (3.6 g/m2)/simustine (250 mg/m2) plus ATG (modified Bu/Cy plus ATG) as preparative therapy in T cell–replete haploidentical hematopoietic stem cell transplantation (haplo-HSCT) for acute leukemia. From August 2009 to August 2013, 38 consecutive patients using TBI/Cy plus ATG regimen for T cell–replete haplo-HSCT (TBI group) at our center were eligible, which contained 28 high-risk and 10 standard-risk patients. A nested case-control study was designed. Seventy-seven patients using modified Bu/Cy plus ATG regimen (Bu group) were randomly selected in a 1 to 3:1 ratio matching for age, disease and status, year of HSCT (±2 years), and length of follow-up. Only 1 graft failure occurred in the TBI group. The incidence and time of neutrophil and platelet engraftment were comparable between the 2 groups. Severe grades III/IV graft-versus-host disease was observed in 13.4% of Bu group and only 2.6% of TBI group (P = .083). More toxicity of the liver (37.7% versus 10.5%; P = .002) and more hemorrhagic cystitis occurred in the Bu group (49.3% versus 23.7%, P = .008). Diarrhea was more common in the TBI group (44.7% versus 22.1%; P = .031). No significant differences were found in the 2-year incidences of relapse (26.5% for TBI group versus 32.3% for Bu group, P = .742), 1-year transplant-related mortality (12.6% versus 16.2%, P = .862), 2-year overall survival (60.2% versus 57.0%, P = .937), and 2-year incidence of disease-free survival (57.9% versus 56.6%, P = .845) between the 2 groups. We conclude that the TBI/Cy plus ATG regimen seems to be feasible in T cell–replete haplo-HSCT, which promotes stable engraftment and a lower incidence of liver toxicity and hemorrhagic cystitis. However, longer follow-up is necessary to determine the late relapse rate and late toxicity.

Cerebral Vascular Abnormalities in Pediatric Patients With Sickle Cell Disease After Hematopoietic Cell Transplant

Stroke is a common sequela of sickle cell disease (SCD). Patients with SCD who undergo hematopoietic stem cell transplantation (HSCT) with successful engraftment will not experience sickling. This ameliorates one aspect of stroke risk; however, the significance of preexisting cerebrovascular abnormalities remains unclear. We performed a literature search for neurological outcomes following HSCT for SCD. We searched for relevant neuroimaging and neurosurgical protocols. We identified 4 unique studies encompassing 196 patients. Of these, 81 had a history of a stroke, transient ischemic attack (TIA), cognitive dysfunction or cerebrovascular abnormalities identified by pretransplant neuroimaging, achieved stable engraftment, and had long-term follow-up. Of the 81 patients, 1 had peritransplant (10 days prior transplant to 50 days posttransplant) TIA. One had posttransplant TIA within 36 to 72 months. None had strokes. Forty-five underwent cerebral imaging at nonuniform intervals. Among this group, 32 (71%) had stable cerebrovascular abnormalities on imaging, 6 (13%) had improvement, and 7 (16%) showed worsening. Cerebrovascular abnormalities identified on neuroimaging may stabilize, improve, or worsen in patients after successful HSCT. Some patients may have neurological events such as TIA. Neurological outcomes in children with SCD post-HSCT have been inadequately studied.

Naturally acquired microchimerism: implications for transplantation outcome and novel methodologies for detection.

Microchimerism represents a condition where one individual harbors genetically distinct cell populations, and the chimeric population constitutes <1% of the total number of cells. The most common natural source of microchimerism is pregnancy. The reciprocal cell exchange between a mother and her child often leads to the stable engraftment of hematopoietic and non-hematopoietic stem cells in both parties. Interaction between cells from the mother and those from the child may result in maternal immune cells becoming sensitized to inherited paternal alloantigens of the child, which are not expressed by the mother herself. Vice versa, immune cells of the child may become sensitized toward the non-inherited maternal alloantigens of the mother. The extent of microchimerism, its anatomical location, and the sensitivity of the techniques used for detecting its presence collectively determine whether microchimerism can be detected in an individual. In this review, we focus on the clinical consequences of microchimerism in solid organ and hematopoietic stem cell transplantation, and propose concepts derived from data of epidemiologic studies. Next, we elaborate on the latest molecular methodology, including digital PCR, for determining in a reliable and sensitive way the extent of microchimerism. For the first time, tools have become available to isolate viable chimeric cells from a host background, so that the challenges of establishing the biologic mechanisms and function of these cells may finally be tackled.

Double Umbilical Cord Blood Transplantation Offers Stable Donor Engraftment and The Prospect Of Cure In Adult Patients With High-Risk Hematologic Malignancies

Allogeneic stem cell transplantation (allo-SCT) remains the main therapeutic option for patients with high-risk hematologic malignancies, albeit with the requirement of a properly matched and timely available donor. Dual-unit umbilical cord blood transplantation (dUCBT) has become an alternative modality, which offers immediate access to allo-SCT for most adult patients who lack an appropriate volunteer donor.We retrospectively analyzed the outcomes of consecutive dUCBT procedures that were undertaken by our center over a seven-year period, with focus on factors affecting engraftment and survival.Between 2006 and 2013, 40 patients underwent dUCBT at a median age of 37 years (range, 16-60) for various hematologic malignancies (acute myeloid leukemia: 22, myelodysplastic syndrome: 5, chronic myelogenous leukemia: 2, acute lymphoblastic leukemia: 6, mixed-phenotype acute leukemia: 2, plasmacytoid dendritic cell neoplasm: 1, hepatosplenic T cell lymphoma: 1, chronic lymphocytic leukemia: 1). The majority of patients (73.7%) had advanced or intermediate-phase disease at the time of transplantation, with a median time from diagnosis to transplant of 17.7 months (range:3.1-92.3). Recipient body weight ranged from 48 to 110 kg (median, 73). The conditioning regimen was myeloablative in 33 (82.5%) patients (busulfan-based in 22, and total body irradiation-based in 11 cases). Antithymocyte globulin was not administered during conditioning, with the exception of one case.Most units (55/80, 68.75%) were 4/6 antigen matched to recipient at HLA-A, -B, and -DRB1 loci, and the remaining were 5/6 matched. By retrospective high-resolution typing for class I HLA alleles, histocompatibility was demoted in 62.3% of units. By additional allele-level typing at HLA-C and -DQB1 loci, the degree of compatibility varied from 8/10 to 3/10, with 80.5% of the units being ≤6/10 matched to the patient. The median dose of cryopreserved total nucleated cells (TNC) per unit was 2.53 x 107/kg (range, 1.09-5.66). At infusion, patients received in total a median of 4.55 x 107 TNC/kg (range, 2.65-9.3) and 1.7 x 105CD34+ cells/kg (range, 0.54-5.14) from both units.The cumulative incidence (CI) of neutrophil engraftment was 92.5% (37/40 patients), with achievement of an absolute neutrophil count (ANC) greater than 500/uL at a median of 20 days (range, 12-52) (Figure 1). Platelet recovery (>50x109/L) occurred at a CI of 63.2%, and a median time of 84 days (range, 32-363). No influence of cell dose (TNC or CD34+) or of the degree of HLA match on the incidence and kinetics of engraftment could be detected. Acute graft-versus-host disease (aGVHD) of grades II-IV and III-IV developed in 85% and 12.65% of patients, respectively. The CI of chronic GVHD was 31% (extensive in 54.5% of cases). There was a statistical trend for increased incidence of cGVHD with <6/10 HLA match at the allele level (p=0.068; HR, 3.35; 95% confidence interval [ci], 0.92-12.24). Non-relapse mortality (NRM) reached 43.1% (95% ci, 27.0-58.2) at 10.3 months, but no case of NRM was noted thereafter (Figure 2). Major causes of NRM were infection/sepsis (n=11), GVHD (n=3), and engraftment failure (n=3). The CI of relapse was 22.7% (95% ci, 10.7-37.5). Relapse was the cause of death of 6 patients. With a median follow-up of 30 months (range, 2-84), overall (OS) and disease-free survival (DFS) rates at 2 years were 36.5% (95% ci, 21-52) and 34.2% (95% ci, 19.3-49.6), respectively (Figure 3). Sixteen of 40 patients are alive and disease-free for a median time of 30 months from transplant. Age ≤37 years, recipient CMV seronegativity, and early disease phase at transplant were associated with improved OS in univariate analysis. Age remained as the only independent risk factor for OS in multivariate analysis of OS (p=0.022). Age ≤37 years was also found to be associated with reduced NRM (p=0.055), and favorable DFS (p=0.04). [Display omitted] [Display omitted] [Display omitted] In conclusion, dUCBT can lead to stable donor engraftment even across multiple HLA disparities and can overcome the barrier of cell dose. Despite considerable early mortality, dUCBT offers the possibility of long-term survival in about one third of adult patients with poor-prognosis hematologic malignancies, for whom allo-SCT would not be otherwise feasible. Disclosures:No relevant conflicts of interest to declare.

Comparison Of Different Pretransplant Predictive Scores In Patients With Refractory Acute Myeloid Leukemia After Allogeneic Stem Cell Transplantation Including Highdose Melphalan: Results Of a Double-Center Observational Study

Patients (pts) with refractory acute myeloid leukaemia (AML) have a particularly dismal prognosis using conventional supportive therapy and chemotherapy. In this double-center retrospective observational study, sequential high-dose melphalan (HD-Mel) as part of the conditioning regimen for allogeneic hematopoietic stem cell transplantation (aSCT) was administered in 162 adult pts (median age 55, range 17 to 71 years) with overt refractory AML. For every patient pretransplant assessment of transplantation (PAM) score, European BMT (EBMT) score, and Hematopoietic Cell Transplantation-Comorbidity Index (HCT-CI) was calculated. Furthermore, by adjusting the threshold of 30 points PAM score was stratified into low and high risk group. 107 pts (66%) underwent a total body irradiation (TBI) based conditioning regimen and 55 pts (34%) a chemotherapy based regimen after a median interval of 5 days (range 1-18 days) following HD-Mel application. Dose of melphalan was 140 mg/m2 in 150 pts (93%), and 200 mg/m2 in 12 pts (7%). 48 pts (30%) were transplanted with an identical sibling donor (ISD), and 114 pts (70%) with matched unrelated donors (MUD). Pts had mostly intermediate (51%) or adverse (46%) cytogenetic risk subgroups. In 150 pts (93%) a stable engraftment was observed and a complete chimerism could be detected in 124 pts (83%). Acute GvHD grades 0-I developed in 98 pts (60%), whereas higher grades II-IV occurred in 64 pts (40%). The median calculated PAM score was 31 (range 22-89). The corresponding median overall probability of death within 2 years after transplant was 70 % (range 44-95%). Median HCT-CI was calculated with 2 (range 0-9) characterizing a low risk comorbidity. Finally the European BMT (EBMT) score was calculated with more than 5 (range 3-7) in 166 pts (72%) transplanted in this cohort. The cumulative incidence of treatment failure estimate was 68% (95%-CL: 60-76%) after transplant resulting in a cumulative relapse estimate of 35% (95%-CL: 28-43%), and an overall non-relapse mortality (NRM) of 38% (95%-CL: 30-46%). After a median follow-up of 35 months after aSCT among surviving pts, the 3-year overall survival estimate (OS) was calculated 37% (95%-CL: 35-45%), and 5-year OS 30% (95%-CL: 25-40%). Time-dependent multivariate analysis on the primary endpoints OS, relapse-free survival (RFS), relapse incidence, and NRM demonstrated an influence of chronic GvHD on EFS (hazard ratio [HR] 0.443, p=0.0003), NRM ([HR] 0.369, p=0.0032), and relapse incidence ([HR] 0.554, p=0.0467). Furthermore, MUD donors tended to have a favourable impact on relapse risk ([HR] 0.590, p=0.0595) compared to sibling donors. PAM score demonstrated significant influence on OS (p=0.0381) and RFS (p=0.0121). For low and high risk groups stratified PAM score was shown to have significant influence on OS ([HR] 1.175, p=0.0056), RFS ([HR] 1.003, p=0.0160), and NRM ([HR] 1.283, p=0.0049), respectively. No influence could be observed for EMBT score and HCT-CI on none of the primary endpoints. In addition the factors age and cGvHD were found to have significant influence on relapse incidence (p=0.011, and p=0.0051) in multivariate analysis. The results of this large study confirm an association between PAM score risk stratification and outcome after aSCT following sequential conditioning regimen with HD-Mel in patients with refractory AML. We could show, that by using a stratified PAM score with the threshold of 30 points a separation between prognostic risk groups is possible. We can summarize that this protocol is a feasible therapy option for patients with refractory AML. Disclosures: No relevant conflicts of interest to declare.

The Use Of a Novel Nonmyeloablative Conditioning Regimen and Sirolimus For GvHD Prophylaxis To Transplant Patients With Chronic Granulomatous Disease

Chronic Granulomatous Disease (CGD) results from a mutation in the NADPH oxidase complex. As a result, patients are prone to recurrent infections and an increased risk of autoimmune disorders such as colitis. Currently, the only available cure is hematopoietic stem cell transplantation using a related or unrelated donor. In 2002, the NIH published their results using a nonmyeloablative regimen and sibling matched donors. Although the results were overall promising, there was still significant GvHD in older patients and a number of graft rejections in the younger patients. Further accrual was limited due to donor unavailability.In 2007, after establishing an agreement with the National Marrow Donor Program, we were able to initiate a protocol for patients with primary immunodeficiencies using an HLA matched unrelated donor. The goal of this protocol was to achieve engraftment in patients with CGD, including high risk patients due to the presence of an ongoing infection or inflammation, without increasing the rate of graft versus host disease. We therefore devised a novel conditioning regimen of Busulfan, Campath, and TBI along with sirolimus as the sole GVHD prophylaxis.To date we have transplanted 21 evaluable patients. Results are summarized below:AgeInfectionInflammationaGvHDcGvHDadditional cells?A&Wcause of death32XX1Nrefused dialysis25XGrade 1Y21XXGrade 1Y19X (colostomy)Grade 4XNinfection, GvHD of skin17XX2NEvan's/TRALI/GvHD17XNpulmonary hemorrhage17XGrade 2Y12XlimitedXY7XY8XY8X (colostomy)Grade 1Y8XXY6XX3NGvHD after 3rd transplant5XY4XY4XXGrade 2Y17Y11Grade 1Y10Y10Y8Y1. Received peripheral blood stem cells from same donor after receiving bone marrow2. Received cells after additional conditioning in the setting of Evan's syndrome3. Received additional cells with initial graft failure. Went on to a second then third transplant with a different conditioning regimen and different donor.Overall survival was 76%; however all deaths occurred in high risk patients and 2 of the 5 were unrelated to the initial transplant. Further, all surviving patients transplanted with high risk disease (11 of the 16) continue to have stable engraftment and had complete resolution of their inflammation and/or infection. This includes a patient with P40phox deficiency whose primary manifestation of CGD was colitis as well as a patient with an invasive fungal infection requiring emergency laminectomy 3 weeks prior to transplant.We have had limited GvHD to date and this occurred primarily in the high risk patients (6 of the 7). The most severe GvHD occurred in a patient given additional cells due possible poor engraftment and persistent thrombocytopenia. In retrospect, this may have been a sign of GvHD and not graft failure; however the result was severe GvHD of the skin and ultimately death from sepsis. Further, the only chronic GvHD (transient, now resolved) was also in a patient given additional cells for concerns of possible graft failure. Subsequently, the protocol was modified to no longer give additional unmanipulated cells and no graft failures or any severe GvHD has occurred in any of the subsequent patients.In general, patients tolerated the transplant well, although we did see a higher than expected rate of engraftment syndrome, again in the high risk patients only (5 of 21). Many patients needed only 1 or 2 transfusions of either platelets or red blood cells and 3 did not require any transfusions at all. We also transplanted two patients with CGD/McLeod's, banking autologous blood prior to the transplant, and only one patient required any blood (1 autologous unit). Three patients did require multiple infusions due to prolonged time to engraftment or slow platelet recovery including the one patient to receive bone marrow as their initial donor product. For the one patient with late graft failure, there was autologous recovery.Thus in this single centre study we have transplanted 21 patients to date including 16 of those considered high risk using a novel non-myeloablative transplant regimen and an unrelated donor. We have had significantly lower rates of GvHD (33%) of which <10% was greater than Grade 2 and only one patient with graft failure. Overall, the combination of Campath and Sirolimus, along with Busulfan and low dose radiation is well tolerated and has a low risk of graft versus host disease while still allowing engraftment in patients, even those with high risk disease. Disclosures:No relevant conflicts of interest to declare.

BCR-ABL1+ Leukemic Stem Cells Are Dependent On Selectin-Ligand Interactions For Engraftment In The Bone Marrow Niche

Leukemic progenitors from patients with chronic myelogenous leukemia (CML) have defects in the adhesive function of β1 integrins and in their response to the chemokine CXCL12 (Stem Cells 2002;3:259), pathways that are critical for engraftment and maintenance of normal hematopoietic stem cells (HSC) in the bone marrow (BM) niche. Previous studies in a mouse BCR-ABL1 retroviral transduction/transplantation model of CML demonstrated that BCR-ABL1+ leukemic stem cells, but not normal murine HSC, are dependent on expression of the CD44 adhesion molecule for BM homing and engraftment (Nat Med 2006;12:1175). To investigate further other adhesion molecules required for stable engraftment of CML stem cells, we employed donor and recipient mice with targeted mutations in genes encoding selectins and those required for expression of selectin ligands in the mouse retroviral CML model. Neither the β1 integrin ligand VCAM-1 nor P-selectin was required in the BM endothelium of recipient mice for efficient engraftment and induction of CML-like myeloproliferative neoplasia (MPN) by BCR-ABL1-expressing stem cells. By contrast, loss of recipient E-selectin significantly impaired engraftment of BCR-ABL1+ stem/progenitor cells, as demonstrated by decreased frequency of BM proviral leukemia-initiating clones, a defect overcome by direct intrafemoral injection of the leukemic cells. We also demonstrated a requirement for expression of selectin ligands on the leukemic stem cells, as BCR-ABL1-expressing stem cells lacking enzymes contributing to biosynthesis of selectin ligands (Core2 or Fucosyltransferases IV and VII) exhibited decreased efficiency of engraftment, resulting in attenuated disease. Further, donor cells deficient in P-selectin glycoprotein ligand-1 (PSGL-1) or in both PSGL-1 and CD44, a glycoform of which can function as a selectin ligand on hematopoietic stem cells (J. Biol Chem 2001;278:47623), displayed intermediate or profound impairment in engraftment of CML-like leukemia, respectively. Removal of selectin ligands by neuraminidase treatment of BCR-ABL1-transduced HSC blocked engraftment of leukemic stem cells and resulted in long-term survival of transplant recipients. Together, these results demonstrate that BCR-ABL1-expressing stem cells are dependent on selectin ligands, expressed on PSGL-1 and possibly on CD44, and their interaction with BM E-selectin for engraftment and retention in the BM microenvironment. Disrupting these niche interactions could represent new avenues for targeting CML stem cells that are resistant to ABL kinase inhibitors like imatinib. Disclosures:Van Etten:Bristol Myers Squibb: Consultancy; Deciphera Pharmaceuticals: Consultancy; TEVA Pharmaceuticals: Consultancy, Research Funding.

Post-Transplantation Cyclophosphamide (PT-CY) After Myeloablative Conditioning Regimen (MAC) In Unmanipulated Haploidentical Bone Marrow Transplantation (hBMT) Is Highly Effective As Gvhd Prophylaxis and Disease Free Survival In Pediatric and Adult Patients With High Risk Leukemia

Introduction BMT from HLA haploidentical family members (hBMT), using either T-cell depleted or T-cell replete grafts, is increasingly employed in patients who urgently need (but lack) a donor. After the promising results in hBMT obtained by the Baltimore group (Luznik et al., Biol Blood Marrow Transplant 2008, Blood 2010) introducing post-transplant high-dose cyclophosphamide (PT-CY) following a non-myeloablative conditioning regimen, the Genoa group (Raiola et al., Biol Blood Marrow Transplant 2013) has shown, in the haploidentical setting that a myeloablative conditioning (MAC) with PT-CY (on day +3 and +5), cyclosporine from day zero and mycophenolate mofetil (MMF) from day +1, is associated with a high rate of engraftment, acceptable TRM and GVHD, and induces durable remission with low incidence of relapse in a high proportion of patients with high-risk hematologic malignancies. We now report a series of 13 heavly pretreated high-risk leukemia patients who underwent a MAC protocol consisted of TBF regimen: Thiotepa (10 mg/kg), Busulfan (9,6 mg/kg) and Fludarabine (150 mg/mq). As graft versus host disease (GVHD) prophylaxis, 2 patients received a 5 drugs combination (ATG-Fresenius, CSA, MTX, MMF and Basiliximab) and stem cells source was G-CSF-primed haploidentical bone marrow (hBM) cells (Di Bartolomeo et al., Blood 2013). In 11 patients stem cells source was non-primed unmanipulated hBM cells, and GVHD prophylaxis consisted of PT-CY (50mg/kg) iv on day +3 and +4, CSA 2.5 mg/kg/day and MMF 45 mg/kg/day from day +4. Results From 2008 to 2013, 11 adult patients (M/F=8/3; median age 40 years, range 23 to 62) and 2 pediatric patients (M/F=1/1, age 4 and 7 years) with high risk ALL (1 pediatric; 2 adult), AML (1 pediatric; 8 adults) and one Richter evolution of CLL, underwent T replete hBMT. At time of transplant 10 patients were in CR (CR1=9, CR2=1), 1 in a good PR and 2 had refractory disease. Grafts contained a median of 5.68 x 108/kg nucleated BM cells (range 0.7-62.1), 2.14x106/kg CD34+ cells (range 0.55-7.09) and 34.7x106/kg CD3+ cells (range 16-93.4). 12 patients are evaluable for engraftment. This occurred in 100% for neutrophils and 93% for platelets, with a median time to neutrophil recovery (&gt; 0.5 x 109/L) of 22.5 days (range, 13-40) and to platelet recovery (&gt; 20.0 x 109/L) of 27.5 days (range, 17-49). All patients had full donor chimerism by day +30. Non relapse mortality (NRM) was 23%: 2 pt died for sepsis (one before engraftment), 1 for aGVHD. 4 patients had grade I and 3 grade II aGvHD, respectively. Only one patient had a grade III, and another had grade IV fatal GVHD. 3 patients had only limited chronic GvHD. Up to now, only 3 patients have relapsed and of these 2 have died. 8 patients (61.5%) are currently surviving, 7 of them are disease-free with median follow-up time of 165 days (range 32-896 days) from transplant. Conclusion Our preliminary results confirm that haplo-BMT following a MAC regimen, is a feasible treatment in pediatric and adult patients, and that PT-CY is highly effective as GVHD prophylaxis, producing a high rate of stable engraftment with encouraging non relapse mortality and relapse-free survival. Disclosures: No relevant conflicts of interest to declare.

Intravenous Pentamidine Is Safe and Effective as Primary Pneumocystis Pneumonia Prophylaxis in Children and Adolescents Undergoing Hematopoietic Stem Cell Transplantation

Pneumocystis carinii pneumonia (PCP) is a potentially life-threatening but preventable infection that may occur after hematopoietic stem cell transplantation (HSCT). Intravenous pentamidine has been used in the prevention of PCP in the post-transplant period, although there are few trials published in the literature evaluating its safety and efficacy. We retrospectively reviewed the medical records of children who underwent HSCT from January 1, 2005, to October 1, 2011, who received intravenous pentamidine as first-line PCP prophylaxis initiated at admission. Demographic, clinical, microbiologic, management and outcome data were collected. One hundred sixty-seven consecutive HSCTs in 137 pediatric patients were given intravenous pentamidine before myeloablation and then every 28 days until the subject was at least a minimum 30 days post-HSCT, had stable neutrophil engraftment (absolute neutrophil count >1000/mm for 3 days without growth factor support) and for allogeneic patients, no evidence of active graft versus host disease and weaning on their immunosuppressive therapy. No cases of PCP were seen in this cohort. Ten (7%) had a grade I side effect of nausea/vomiting requiring slower infusion time and 2 (2%) had a grade IV reaction with anaphylaxis (rash) and hypotension with 1 child requiring transfer to the intensive care unit. Intravenous pentamidine was safe and effective for the prevention of PCP in pediatric HSCT patients. Given the potential neutropenic effects of trimethoprim-sulfamethoxazole, compliance with drug administration and inferior efficacy of other PCP prophylactic medications, intravenous pentamidine should be considered as first-line therapy for the prevention of PCP in children undergoing HSCT.

Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model

Background aimsThere is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion. MethodsAn ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays. ResultsIP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion. ConclusionsIP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.

Comparing High and Low Total Body Irradiation Dose Rates for Minimum-Intensity Conditioning of Dogs for Dog Leukocyte Antigen–Identical Bone Marrow Grafts

We tested the hypothesis that total body irradiation (TBI) given at a high dose rate would be more immunosuppressive and lead to a higher incidence of stable hematopoietic cell engraftment after suboptimal levels of conditioning irradiation compared with TBI at a low dose rate. We assessed the engraftment success of dog leukocyte antigen–identical bone marrow transplantation in recipients of 100, 150, and 200 cGy TBI administered at a rate of 7 or 70 cGy/min. Dogs received donor marrow on the same day as TBI and were subsequently treated with postgraft immunosuppression consisting of mycophenolate mofetil (for 28 days) and cyclosporine (for 37 days). Donor chimerism was monitored until the end of study and was characterized by either graft rejection or stable engraftment. Increasing the radiation dose rate from the traditional 7 cGy/min to 70 cGy/min did not lead to increased engraftment success at any of the irradiation doses tested. The dose rate of 70 cGy/minute was no more hematotoxic than the rate of 7 cGy/minute. TBI delivered at a high dose rate was well tolerated but was not associated with a better rate of allogeneic hematopoietic cell engraftment compared with TBI delivered at a lower dose rate.

Severe acute radiation syndrome: treatment of a lethally 60Co-source irradiated accident victim in China with HLA-mismatched peripheral blood stem cell transplantation and mesenchymal stem cells

This is a case report of a 32-year-old man exposed to a total body dose of 14.5 Gy γ-radiation in a lethal 60Co-source irradiation accident in 2008 in China. Frequent nausea, vomiting and marked neutropenia and lymphopenia were observed from 30 min to 45 h after exposure. HLA-mismatched peripheral blood stem cell transplantation combined with infusion of mesenchymal stem cells was used at Day 7. Rapid hematopoietic recovery, stable donor engraftment and healing of radioactive skin ulceration were achieved during Days 18–36. The patient finally developed intestinal obstruction and died of multi-organ failure on Day 62, although intestinal obstruction was successfully released by emergency bowel resection.

Stable human hematopoietic stem cell engraftment supports continuous de novo generation of mature human blood cells in mice

Stable human hematopoietic stem cell engraftment supports continuous de novo generation of mature human blood cells in mice

Haploidentical hematopoietic stem cell transplantation for the treatment of severe aplastic anemia in children

This study was purposed to assess the effectiveness of haploidentical hematopoietic stem cell transplantation (HSCT) without in vitro T cell depletion for the treatment of severe aplastic anemia (SAA) in children. Two children with SAA/very SAA (VSAA) received T cell-depleted HSCT from their fathers with 2 loci mismatched in our center between October 2010 and March 2013. During 4 months after onset, both failed in treatment of cyclosporine (CsA)+ granulocyte colony stimulating factor (G-CSF), had active or serious infections, were transfusion dependent and lacked HLA-identical sibling donors and unrelated donors. The conditioning regimen before HSCT included fludarabine, cyclophosphamide and thymoglobulin. The source of grafts was a combination of G-CSF mobilized peripheral blood hematopoietic stem cells and BM. The recipients received CsA, mycophenolate mofetil (MMF) and short-term MTX for GVHD prophylaxis. Both children with SAA achieved 100% donor myeloid engraftment. Neutrophil engraftment occurred at day 12 and day 18 after transplant respectively. Platelet engraftment occurred at day 17 and day 26 after transplantation respectively. Two patients all developed grade I acute graft versus host disease (GVHD), one of which evolved into chronic limited GVHD well-controlled. Both have survived for two years after transplantation with 100% donor myeloid engraftment and effective lymphoid reconstitution. In conclusion, these limited cases suggest that haploidentical HSCT for children with SAA without a HLA-identical sibling donor and unrelated donor may be feasible. Further prospective clinical study is required to increase the overall survival (OS) by decreasing GVHD while maintaining stable engraftment.