Full Chimerism Research Articles

The Liver Graft as Trojan Horse–Multilineage Donor-Derived Hematopoiesis After Liver Transplantation: Case Report

Hematopoietic macrochimerism, which is rarely seen after orthotopic liver transplantation (OLT), has been linked to the development of graft versus host disease (GvHD). We report on a patient with GvHD after OLT in whom full engraftment of donor-derived, multilineage hematopoiesis occurred, indicating that the liver contains pluripotent hematopoietic progenitor cells (HPC) capable to restore hematopoiesis in recipients. Although preventing graft rejection, standard immunosuppressive therapy may be under certain immunological conditions not sufficient to prevent GvHD. Age-, disease-, and treatment-related variables might be critical determinants for the development of an effective alloreactive T-cell response leading to the establishment of full hematopoietic chimerism.

113-P: TCR REPERTOIRE AS A BIOMARKER FOR CHIMERIC KIDNEY RECIPIENTS BENEFITING FROM FACILITATING CELL-ENRICHED STEM CELL TRANSPLANTATION

In recent publication we reported that durable chimerism and tolerance can be achieved with minimal toxicity and without GVHD in highly-mismatched transplant (Tx) recipients through low intensity conditioning and a living donor kidney Tx with a dose of hematopoietic stem cells (HSC) enriched for facilitating cells (FC) (Sci. Transl. Med. 2012; 4 (124): 124re28). We herein present data evaluating TCR expression as a biomarker for chimeric immune reconstitution in patients with ⩾ 2 years of follow up. Four patients were randomly selected from the study by STR analysis. Two had achieved full chimerism and the other two did not. Peripheral blood samples were freshly obtained from donors and recipients and T-cell subsets were isolated using MACs microbead system. DNA was extracted and sequenced by ImmunoSeq (Adaptive Biotech) to evaluate TCR clonal repertoires in recipients. Even though the clone diversity in TCR repertoire was reduced in post-Tx recipients (0.9 ± 0.05 pre-Tx vs. 0.79 ± 0.09 post-Tx), the repertoire was diverse enough to suggest recovery of immune competence. Interestingly, at least 97% of the unique sequences observed in post-Tx recipient were not present in either donor or recipient pre-Tx. Within the pool of shared sequences, full chimerism correlated with a shift towards homology with the donor, while loss of chimerism with recipient pre-Tx. In addition, the chimeric patients also exhibited reduced diversity of TCR sequences and increased clonality. Top 20 “high frequency” clones are most stably expressed. CD8+ cells had highest number of “high frequency” clones (CD8+ > PBMC > CD127-CD4+CD25- > CD127-CD4+CD25+ Tregs). Notably, the pattern of “low frequency” clones was highest in CD127-CD4+CD25+ Treg subset indicating extensive and rapidly changing TCR repertoire. The HLA-mismatched kidney recipient benefiting from infusion of FC-containing HSC ensure a new and extensive TCR repertoire that partially resembles that of the donor. Ildstad: Regenerex LLC: Other: Chief Scientific Officer.

Allogeneic stem cell transplantation using alemtuzumab-containing regimens in severe aplastic anemia

Alemtuzumab, a humanized anti-CD52, IgG1 monoclonal antibody, is used to reduce graft-versus- host disease (GVHD) and aid engraftment after allogeneic haemopoietic stem cell transplant (HSCT). Its associated low incidence of GVHD makes it an attractive alternative to anti-thymocyte globulin (ATG) in transplant conditioning regimen for severe aplastic anaemia (SAA). We have reviewed the use of alemtuzumab-based conditioning regimen for HSCT in SAA and show that it results in sustained haematological engraftment, a very low incidence of chronic GVHD without an increase in viral infections. Intriguingly, alemtuzumab appears to induce tolerance post-HSCT with the findings of stable mixed T cell chimerism with full donor myeloid chimerism and the absence of chronic GVHD, and which persist on withdrawal of post-graft immunosuppression. Finally, its low toxicity profile may permit future application of HSCT to older patients with SAA who fail to respond to immunosuppressive therapy.

Hematopoietic stem-cell transplantation for sickle cell disease: current evidence and opinions

With rapidly expanding evidence of benefit reported by several groups, allogeneic hematopoietic stem-cell transplantation has become an acceptable treatment option for sickle cell disease. It is currently the only curative therapy available. Hematopoietic stem-cell transplantation was offered primarily as a therapeutic option for severe sickle cell disease in the context of myeloablative matched sibling donor transplants over the last two decades and helped to establish the benefits of transplantation for this disorder. While this approach provided proof of principle, the disadvantages and limitations of transplantation became evident along the way. It has been recognized that transplantation for sickle cell disease does not need to adhere strictly to the principles of transplantation for malignant disorders, such as achievement of full donor cell chimerism. As reviewed here, in recent years the transplant community has set out to explore ways to make stem-cell transplantation more available to patients with the disease, define indications and better timing, and offset toxicities with novel approaches to conditioning and better supportive care.

Intramural Duodenal Haematoma After Upper Gastrointestinal Endoscopic Biopsy in a Bone Marrow Transplant Recipient

s / Biol Blood Marrow Transplant 19 (2013) S279eS312 S302 Fisher's exact test. Disease free (DFS) and overall survival (OS) were estimated using Kaplan-Meier methods, and timeto-relapse and non-relapse mortality (NRM) were estimated using cumulative incidence functions. Cox regression was used to further investigate the association between chimerism and the risk of NRM adjusted for key covariates. Results: Of 161 patients identified, 96 were disease free and had PB TC chimerism at 3 month Table 1 lists transplant characteristics and outcomes. PB TC MC was observed in 64 (66%) at 3 month There was no difference in chimerism based on disease, stem cell source, HLA-match, donorrecipient gender pairing, or conditioning regimen. Relapse and DFS were not different between the MC and full donor chimerism groups. However, NRM was significantly higher (P-value 0.001) and OS was significantly lower (P-value 0.01) in the full donor chimerism group at 2 years. Using Cox regression, the association between chimerism and NRM remained after adjusting for DLI, disease, conditioning regimen and donor-recipient sex (P value 0.03). Analysis of 6 month PB TC chimerism also showed patients withMC in the PB at 6 months had less NRM and improved OS than patients with full donor chimerism. Conclusions: In this analysis of PB TC chimerism following TCD transplant, both NRM and OSwere significantly better in patients with MC. These data suggest that DLI for the conversion of MC to full chimerism may be unnecessary.

Romiplostim resistance in secondary failure of platelet recovery

Romiplostim, a thrombopoietin mimetic, is FDA approved for the treatment of thrombocytopenia in patients with refractory immune thrombocytopenia. Given the success of thrombopoietin agonists in the treatment of immune thrombocytopenia, data in other clinical settings are emerging. In the bone marrow transplant setting, secondary failure of platelet recovery and persistent thrombocytopenia are associated with increased mortality. In this case, romiplostim was attempted in a chronic lymphocytic leukemia patient who underwent a matched, unrelated donor, non-myeloablative stem cell transplant. This patient experienced profound thrombocytopenia despite dose escalation. Anti-thrombopoietin or anti-romiplostim antibodies were not detected by specific assays. Furthermore, the patient remains in disease remission with full chimerism. This case demonstrates that heavily pre-treated patients with limited bone marrow reserve may not respond to thrombopoietin agonists.

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.

Bone Marrow Mixed Chimerism Is a Risk Factor for Relapse After T-Cell Depleted Allogeneic Stem Cell Transplant

AbstractAbstract 4145 Background:Full donor chimerism following non myeloablative unmodified stem cell transplants (SCTs) is important for disease control and SCT outcome. The impact of mixed chimerism (MC) after myeloablative unmodified SCT remains controversial, and there are limited reports about its prognostic significance after T-cell depleted (TCD) SCT. Treatment of MC, including reduction of immunosuppression or infusion of donor leucocytes (DLI), can lead to the development of graft versus host disease (GVHD). In order to optimize the graft vs leukemia (GVL) effect of a TCD allograft while minimizing GVHD risk, we sought to determine the significance of MC on TCD SCT outcomes and to identify a subgroup that would benefit from intervention. Methods:From 2000–2010, 447 adult patients with acute myeloid and lymphocytic leukemia (AML, ALL) and myelodysplastic syndrome (MDS) underwent bone marrow (BM) or peripheral blood stem cell (PBSC) TCD SCT. Conditioning regimens were total body irradiation (TBI)/Thiotepa with Cyclophosphamide or Fludarabine, Clofarabine/Melphalan/Thiotepa or Busulfan/Melphalan/Fludarabine. Like-gender donor-recipient chimerism was analyzed using semi-quantitative PCR for polymorphisms; PCR and/or cytogenetics were used for unlike gender pairings. Relapse was cytogenetic or hematologic evidence of disease. BM chimerism and disease status were evaluated at approimately 6, 12, 18 and 24 months. BM chimerism and outcome data were reviewed to determine whether 6 month BM MC (defined as < 95% donor chimerism) was associated with 2 year post-SCT relapse, transplant-related mortality (TRM) and disease-free survival (DFS). The 6 month landmark was chosen as a reasonable time point for intervention, such as DLI, if needed. Patients who received DLI prior to landmark were excluded. Patient and SCT characteristics were compared using Wilcoxon’s rank-sum test or Fisher’s exact test. DFS was estimated using Kaplan-Meier methods, and time-to-relapse and TRM were estimated using cumulative incidence functions. Results:300 of the 447 patients survived, were disease free, and had BM chimerism data at 6 months. Median age was 50.6 years. Most patients received PBSC grafts (89%). 131 (44%) and 9 (3%) related, and 86 (29%) and 74 (24%) unrelated donors, were matched and mismatched, respectively. Only patients in remission or with low level disease (BM blasts < 10%) were transplanted with TCD grafts. The majority of patients were conditioned with Busulfan/Melfalan/Fludarabine and TBI/Thiotepa/Fludarabine, 45% and 32%, respectively. 251 pts (84%) received antithymocyte globulin. 25% demonstrated MC in the BM at 6 mos. The majority (60%) with MC had a donor component of 75–95%. Diagnosis was not associated with MC at 6 months. Stem cell source and degree of HLA-match had no impact on BM chimerism. There was a significant difference in chimerism based on donor-recipient gender pairing (P = 0.04): male grafts into female patients having the greatest risk of MC and female grafts into female patients having the least. There was also a significant difference in the degree of chimerism based on conditioning regimen: Busulfan/Melphalan/Fludarabine was most likely to result in MC (P = <0.001). TRM was not significantly different between the mixed and full donor chimerism groups. However, relapse was significantly higher in the MC group (P = 0.03): 10% (95% CI: 6–14%) with full chimerism and 18% (95% CI: 7–28%) with MC at 2 years (Figure 1). Diagnosis had no impact on relapse among patients with MC. Conclusions:25% of TCD SCT recipients had MC in the BM. Although the incidence of relapse was low for patients who survived to 6 months in the full and MC groups (10% and 18%, respectively), the difference was statistically significant. There was also a significant difference in incidence of MC based on conditioning regimen and donor-recipient gender pairings. The low percentage of relapse in the MC group precluded subset analyses to identify a group who would benefit from the GVL effect of DLI. Lineage-specific PB chimerism analysis is ongoing and may provide information about subgroups that would benefit from intervention for MC. Analyses of outcomes, i.e., GVHD, in recipients of DLI or TCD stem cell boost for MC may help to determine the risk-benefit ratio of such interventions. [Display omitted] Disclosures:Goldberg:SOBI Biovitrum: Research Funding. Perales:SOBI Biovitrum: Research Funding.

Poor Graft Function in Recipients of T Cell Depleted (TCD) Allogeneic Hematopoietic Stem Cell Transplants (HSCT) Is Mostly Related to Viral Infections and Anti-Viral Therapy.

AbstractAbstract 3147 Introduction:Poor graft function (PGF) without immune rejection, defined as persistent cytopenias with hypocellular marrow and full donor myeloid chimerism, can be a life-threatening complication after allogeneic HSCT. It is commonly caused by viral infectious, myelosuppressive drugs like antivirals, and graft-vs-host disease (GvHD). Treatment options include supportive therapy with transfusions and growth factors and in severe cases administration of additional hematopoietic stem cells (HSCs) from the same donor without conditioning (stem cell boost). The incidence, natural history, and the indications for stem cell boost therapy are not well defined. Aims:To assess the incidence, etiologies, and indications for stem cell boost for PGF in a homogeneous group of patients with advanced MDS and AML who underwent TCD HSCT from matched or mismatched related or unrelated donors after conditioning with the same myeloablative regimen. Patients and methods:Poor graft function was defined as persistent neutropenia (ANC <1,000 μL and G-CSF administration x3 in 30 days), thrombocytopenia (platelets <50,000 μL or platelets transfusion × 4 in 30 days), and/or hemoglobin <8 g//dL after engraftment with hypocellular BM and full donor myeloid chimerism. Severe PGF was defined as ANC <500 μL, red cell transfusion-dependent anemia with reticulocytopenia of < 20,000 μL, and platelets <20,000 μL. The patient population in which this study was done included 42 patients enrolled between 09/2009 and 05/2012 in a phase 2 trial of palifermin peri-transplant to reduce transplant-related mortality. The median age was 57.5 years (1–65). All patients received the same myeloablative conditioning regimen with busulfan, melphalan, fludarabine, rabbit ATG and palifermin peri-transplant. G-CSF mobilized donor peripheral blood stem cells underwent CD34+ selection and depletion of T cells using CliniMACS immunomagnetic selection columns (Milteny Biotec). Donors were HLA matched (31; 13 related and 18 unrelated) or mismatched unrelated (11). Chimerism was determined in bone marrow as well as neutrophils, B cells, and T cells by short tandem repeat analysis on DNA extracted from bone marrow and peripheral blood cell subsets. Results:Forty-one patients were evaluable for this analysis; 1 patient was not included as he rejected the allograft shortly after engraftment. There were 8 cases of PGF with a cumulative incidence (CI) at 1 year of 18% (13% HLA matched, 33% HLA mismatch). The etiology was infection in 7 cases, and unknown in the 8th case. This patient presented with presumed autoimmune anemia and thrombocytopenia associated with a hypercellular marrow and did not respond to multiple lines of therapies. Her marrow became later hypocellular and met the criteria for PGF. None of the PGF cases in this series was associated with GvHD at the time of diagnosis of PGF. The infectious etiologies included: 6 viral infections and 1bacterial sepsis + myelosuppressive drugs. The most common viral etiology associated with PGF was CMV (50%). The 1-year CI of PGF in CMV seropositive patients was 25% and in CMV seronegative patients was 14%. Of note, HHV6 viremia was detected in patients with PGF. HHV6 is not routinely monitored, however, making it difficult to establish a causative role. All patients had moderate PGF at diagnosis and 3 cases had worsening of cytopenias and met the criteria for severe PGF. To date, 3 PGF patients have died from EBV-PTLD, adenovirus infection or GVHD (developed after CMV treatment with liposomal cidofovir), 3 continue to suffer from PGF and 2 patients are alive with recovered good blood counts after eradication of CMV. Of the 3 patients with persistent PGF, one received a TCD boost with no response, and 2 continued to be treated for CMV viremia. A stem cell boost was indicated if pancytopenia persisted despite eradication of cause of the PGF. In this small series, there were not enough events to evaluate association between PGF and CD34 cell dose, CD3 cell dose or day 100 T-cell chimerism. Conclusions:In this homogenous population of patients with MDS who underwent TCD allogeneic HSCT, the incidence of PGF is about 20%. The most common cause was viral infection with predominance of CMV. Therefore, strategies to prevent CMV reactivation in patients undergoing allogeneic HSCT has the potential to reduce the risk of PGF and avoid the need for infusion of additional stem cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Engraftment Kinetics After Alimtuzumab-Based Allogeneic Stem Cell Transplant (SCT): Full Donor T-Cell Chimerism Did Not Predict for Acute Graft Versus Host Disease (GVHD) or CMV Reactivation

Abstract 4162 Background:Graft versus host disease (GVHD) remains the leading cause of the treatment failures following an otherwise successful allogeneic Stem Cell Transplant. Alemtuzumab – based prophylactic regimens is evolving as an important option for prevention of this devastating complication. Multiple phase II studies suggested that patients who received Alemtuzumab –containing GHVD prophylactic regimens tends to have less acute GVHD though the utility is limited by increased incidence of mixed chimerism often requiring donor lymphocytes infusion and cytomegalovirus reactivation. Hypothesis:We hypothesize that if we could identify an optimal dose of Alemtuzumab; we could maintain the benefit of lowering the incidence of GVHD and also minimize mixed chimerism and CMV reactivation following the transplant. Method:To test our hypothesis, we conducted a pilot study utilizing the combination of Alemtuzumab (20 Related Donor: 30 mg on Day -1; 10 Unrelated donor: 30 mg daily on Day -2 & Day -1) and cyclosporine as GVHD prophylaxis. Between 5/11 and 5/12, 28 pts were included in the study. There were 15 females and 13 males. The diagnosis included 16 AML, 3 NHL, 2 MDS, 1 CML, 1 CLL, 1 ALL, 1 HD, 1 PLL, 1 MPD and 1 MM. Conditioning regimens consisted of Fludarabine/Melphalan in 20, Fludarabine/Busulfan in 5, TBI/Cy in 2 and Fludarabine/Clofarabine in 1. Upon achievement of engraftment, we monitor CMV by PCR weekly and engraftment by Short tandem repeat (STR) monthly with T-cell and Myeloid subsets analysis. Results:26/28 pts achieved neutrophils engraftment except 1 died shortly after SCT from heart failure and 1 experienced primary graft failure with day 30 STR showing only 3% donor T-cell chimerism. On Day 30 post-SCT, all evaluable pts had predominately donor T-cell (median 99%; 19/27 (70%) ≥ 99%; range 82 to > 99%) and Myeloid Chimerism (24/26 (92%) ≥ 99% except one pt who developed rapidly progressive leukemia phase PTLC after transplant. 25 pts (89%) survived beyond Day 90 post-SCT and have engraftment data available for analysis. On Day 90 analysis, while most pts maintain predominant donor myeloid chimerism (> 95%; median 99%); only 10/25 (40%) had maintained 99% donor T-cell chimerism. In addition, 76% pts had their T-cell donor chimerism worsened by Day 90 when compared with Day 30 analysis (9 pts dropped below 80%; range 8–75%). On the other hand, no pt developed secondary graft failure. 5 pts (17%) developed acute GVHD with only 1 (4%) Grade IV aGVHD. 8 pts (29%) developed CMV reactivation within day 100 post-SCT. Full donor T-cell chimerism did not predict for GVHD or CMV reactivation. With longer follow up, some pts’ have donor T-Cell chimerism increased with tapering of immunosuppressive therapy with or without GVHD or after receiving DLI. Conclusion:1. Our preliminary data suggested that using a lower dose of Alemtuzumab (30 mg for RD and 60 mg for MUD) as GVHD prophylaxis is effective to prevent GVHD though still associated with high incidence of CMV reactivation. 2. Most pts achieve predominant Donor T cell chimerism on day 30 though many worsen on subsequent follow-up. 3. Although it appears that there's no impact on durability of engraftment, longer follow-up is required to determine the impact on relapse, chronic GVHD and survival. Disclosures:Fung:Genzyme: Honoraria. Off Label Use: Alemtuzumab for GVHD prophylaxis.

Short Course of Levofloxacin During Neutropenia Prevents Early and Late Bacteremia Episodes After Allogeneic Blood and Marrow Transplantation (alloBMT)

Abstract 4141Bacteremia is a common complication after alloBMT due to neutropenia, mucositis, GvHD, immunosuppressive agents, and slower immune reconstitution and full chimerism after reduced intensity conditioning. Since symptoms and signs of bacteremia (e.g., fever) may be blunted or absent in BMT recipients receiving intensive immunosuppression, our clinical practice applies a low threshold for obtaining blood cultures. Our goals were to define risk factors for bacteremia at pre-specified periods after BMT and to compare rates of bacteremia using microbiological criteria vs. CDC/NHSN criteria which require symptoms and signs such as fever, chills or hypotension. We performed a retrospective cohort study of 168 adult (>18 yrs) consecutive first alloBMT patients from January 2008 to May 2012. A bacteremia episode (BE) was defined according to the EBMT Infectious Disease Working Party criteria which excludes skin flora contaminants. A second set of positive blood cultures of the same bacterial strain >30 days apart was considered a recurrent BE. Blood cultures which grew different bacterial strains were defined as a single episode of polymicrobial bacteremia if drawn < 48 hrs apart and as unique BEs if drawn > 48 hrs apart. Patient characteristics include: median (range) age 54 (19–73) yrs, 57% male, 49% AML, 14% ALL, 11% MDS, 7% NHL, 20% other diseases, 77% KPS 50–80, 81% peripheral blood, 64% unrelated donor, 23% <10/10 HLA match, 73% reduced intensity conditioning regimens. After 8/2011, all patients received levofloxacin prophylaxis during neutropenia, whereas before 8/2011, no routine antibacterial prophylaxis was used. At discharge, penicillin prophylaxis was initiated for all patients until at least 1 yr post-alloBMT. All patients received voriconazole or micafungin as anti-fungal prophylaxis from day -1 until off immunosuppression and acyclovir as anti-viral prophylaxis for at least 1 yr post-alloBMT. Overall, there were 238 BEs in 168 patients for a rate of 1.42 BE/pt. The rate of BEs was highest in the early peri-BMT period and dropped significantly after day+30 (Table), and 80% of BEs before day+30 occurred during neutropenia. The proportions of patients with >1 BE in each time period were 49%, 31%, and 28%, respectively. While 67% of BEs (defined by microbiological criteria), from admission (first day of conditioning regimen) to day+30 met CDC/NHSN criteria, only 25% of BEs after day+30 did so. The majority of BEs did not result in a central line replacement. From admission through 1 yr post-allo BMT, 59 (35%), 49 (29%) and 60 (36%) patients had 0, 1, and >1 cumulative total BEs, respectively. We performed a univariate analysis of risk factors including gender, age, KPS, diagnosis, donor relation, HLA match, SC type, use of levofloxacin as antimicrobial prophylaxis, and conditioning regimen intensity, for >1 BE for each of the 3 time periods, and cumulatively. Lack of levofloxacin prophylaxis was a significant risk factor for BEs in all time periods post-alloBMT. In addition, grade II-IV acute GvHD was a significant risk factor for >1 or >2 BEs in the day+31 to 100, day+101 to 365 and admission to day+365 time periods. Levofloxacin prophylaxis decreased the rate of patients with >1 BE from 54% to 14% in the day+30 time period (P<0.0001) and from 69% to 36% from admission to 1 yr post-alloBMT (P=0.003). BEs resulted in death within 7 days of first positive culture in 4 patients due to MRSA, E faecium, K pneumonia, and E faecalis/Candida. We observed the greatest discordance between applying microbiological criteria vs. CDC criteria to define BEs from days+31 to 365. This discordance reflects the lack of sensitivity of typical clinical signs/symptoms of infection in the setting of GvHD post-alloBMT, and points to the need for modified criteria to define BEs for this patient population. Development of improved prophylaxis strategies that focus on patients with GvHD may diminish the incidence and severity of these infections.Time Period# of BEs# pts with >=1 BE/# pts evaluable (%)# (%) of BEs meeting CDC criteria# (%) of BEs resulting in a CVC line changeTotal # of Observation Days# BEs/Observation DaysRate of BE/1000 pt-DaysAdmission to Day +309282 (49)62 (67)7 (8)5,9700.015415Day +31 to 1007051 (31)18 (26)10 (14)10,6930.00657Day +101 to 3657640 (28)18 (24)5 (7)29,8360.00253 Disclosures:Off Label Use: Levofloxacin is approved for treatment of infections. We will discuss its use as infection prophylaxis.

Early Clinical Experience of aAPC Activated NK-DLI Following Allogeneic PBSCT in Pediatric Solid Tumor Patients.

AbstractAbstract 3013 Background:Allogeneic Hematopoietic Stem Cell Transplant (alloHSCT) is effective in some hematologic malignancies and studies of alloHSCT for ultra high-risk pediatric solid tumors have shown some promise. Preclinical data demonstrates that activated NK cells readily kill pediatric solid tumors and leukemias, large numbers of activated NK cells can be generated ex vivo using artificial APCs (aAPCs) and the post-transplant period may be favorable for expansion and survival of adoptively transferred NK cells. Methods:We initiated a Phase I trial to assess feasibility and toxicity of escalating doses of donor-derived activated NK cell donor lymphocyte infusions (NK-DLI) on days 7 and 35 days following HLA-matched T cell depleted PBSCT in children and young adults with ultra-high risk solid tumors and leukemias. Donors underwent a single apheresis for filgrastim mobilized PBSC. The product was T cell depleted and CD34 and CD56 selected prior to cryopreservation, and the CD56+ fraction was cultured for 9–11 days with a K562 based aAPC expressing 4-1BBL and IL-15Ra plus rhIL-15 to generate the NK-DLI. NK-DLI consistently expressed high levels of natural cytotoxicity receptors NKp44 and NKp46 and mediated potent cytotoxicity ex vivo. T cell addback to the CD34 selected graft was performed to administer a T cell dose of 0.8–1.4 × 10e4 T cells/kg. Except for two pilot patients where NK-DLI was infused following engraftment, NK-DLI were administered ∼Day 7 and 35 post-transplant. Patients received a reduced intensity preparative regimen (fludarabine 30 mg/m2 and cyclophosphamide, 1200 mg/m2 on days –6 to -3; and melphalan 100 mg/m2 on day -2), and no prophylactic immunosuppression. Results:Seven patients with sarcomas were transplanted with NK-DLI infusion (1×10e5 CD56+ cells/kg/dose). Engraftment was brisk (median platelet recovery at 8d and neutrophil recovery at 9d). Patients had full donor myeloid chimerism by day 14 and all but one had >80% lymphoid chimerism by day 28. Patient #1 received NK-DLI on day +24 following alloHSCT and experienced aGVHD within 24 hours of NK-DLI with bullous skin rash and voluminous diarrhea. Prior to infusion the patient had what appeared to be a viral rash and fevers (skin biopsy negative for GVHD), but in retrospect likely GVHD, which was exacerbated by NK-DLI. Patient #2 received NK-DLI on Day +15 with no adverse effects. Subsequent patients enrolled onto cohort 1 with planned NK-DLI at days 7 and 35 (+7). Two patients developed aGVHD (rash and diarrhea), one with a cytokine-type reaction (fevers, hypotension) within 48 hours of NK-DLI infusion and one with aGVHD starting 21 days post NK-DLI. All 3 patients with matched unrelated donors experienced aGVHD after NK-DLI, whereas the 5 patients with matched sibling donors had no apparent adverse reactions nor GVHD. Persistence/engraftment of infused NK-DLI cannot be definitively determined, however, mean NK cell counts measured pre-alloHSCT and day +28 are 165/mm3 and 668/mm3 in the present cohort (n=6) compared to 149/mm3 and 395/mm3 in a historical alloHSCT population that did not receive NK-DLI (n=24). Antitumor effects via PET imaging were observed in patients with measurable disease and those without disease at the time of transplant have remained NED. Conclusions:Ex-vivo, aAPC expanded NK-DLIs do not interfere with stem cell engraftment but may contribute to or induce aGVHD, particularly in patients with matched, unrelated donors. Preliminary results suggest that aAPC NK-DLI expand in vivo and mediate antitumor effects following allogeneic PBSCT.Table:PatientAge/GenderDiseaseNK Cell DoseDay of NK Infusion (post-HSCT)DonorLikely NK Related ToxicitiesAnti-tumor EffectsStatusPre-HSCT NK CountPost-HSCT/NK1 Count (day 28-35)134 y FDSRCT1 × 105/kgDay 24MUDGrade IV GVHDPET and CT responseDoD-day + 190861883233 y MEwing's1 × 105/kgDay 14MSDNonePET responseDoD-day + 17122257322 y MDSRCT1 × 105/kgDay 9MUDFever, Grade II GVHDCRNED298762416 y MEwing's1 × 105/kgDay 8, Day 33MUDFever, Grade III GVHDRemains NEDNED179180524 y MDSRCT1 × 105/kgDay 8, Day 35MSDNonePR/CRMRD/NED119428618 y MARMS1 × 105/kgDay 8, Day 36MSDNoneRemains NEDNED85698714 y MEwing's1 × 105/kgtoo earlyMSDtoo earlytoo earlyNEDMEAN165668 Disclosures:No relevant conflicts of interest to declare.

Acute GVHD is a strong predictor of full donor CD3+ T cell chimerism after reduced intensity conditioning allogeneic stem cell transplantation

The monitoring of chimerism is a standard procedure to assess engraftment and achievement of full donor lymphoid cells after reduced intensity conditioning (RIC) stem cell transplantation (Allo-SCT). However, there is no consensus on when and how often to monitor post-transplant chimerism. We retrospectively analyzed our experience regarding the impact of acute graft versus host disease (GVHD) for the prediction of allograft chimerism. One-hundred-and-fifteen patients transplanted between 2001 and 2010 were identified. This group included 57 females and 58 males with a median age of 50 years (range: 26-68). Patients evaluated in this study were adult patients with hematologic malignancies, who received transplants from an HLA-matched sibling donor or matched unrelated donor (MUD) at allele level so-called 10/10, and received the RIC regimen including fludarabine/busulfan and anti-thymoglobulin (ATG). Mixed T-cell chimerism was defined as between 5 and 94% recipient cells, and full chimerism was defined as the presence of more than 95% donor T-cell chimerism (TCC). Full donor TCC was achieved in 93 patients (81%) at a median of 77 days (range: 30-120) post-transplant. The cumulative incidence of Grade 2-4 GVHD in our population was 25% (95% CI 17-34). The analysis of the population of patients with acute GVHD grade ≥2 showed that at day 120 after Allo-SCT they all had a total full donor TCC. On the other hand, 78 (68%) patients without acute GVHD grade ≥2 presented with mixed chimerism (p = 0.002) on day 120 post-transplant. Interestingly, patients who received ATG 5 mg/kg obtained a higher probability of complete chimerism compared with those receiving 2.5 mg/kg (p = 0.03). In conclusion, our study demonstrates that acute GVHD was predictive of full donor TCC after RIC Allo-SCT. Therefore, our data may challenge the concept of the frequent or close monitoring of donor chimerism in some patients with ongoing acute GVHD. However, chimerism testing could represent an attractive modality for minimal residual disease detection or for impeding relapse warranting further prospective studies.

Lenalidomide plus donor-lymphocytes infusion after allogeneic stem-cell transplantation with reduced-intensity conditioning in patients with high-risk multiple myeloma

Myeloma relapse is the main cause of death after allogeneic stem cell transplantation. The aim of our observational study was to evaluate the anti-myeloma effect of lenalidomide followed by donor-lymphocyte infusion (DLI) as post-transplantation adoptive immunotherapy. Twelve patients with refractory myeloma were analyzed. The median age at transplantation was 56 years (range, 46-64 years). All patients received reduced-intensity conditioning. Patients were included if progressive or residual disease was observed at day +100 and if no signs of graft-vs-host disease were evident. DLIs were administered after two cycles of lenalidomide. Median dose of lenalidomide was 15 mg (range, 10-25 mg). Patients received a median of six cycles (range, 1-10 cycles). Nine patients (60%) received an escalating dose of DLI. The 1 and 2-year probability of progression-free survival was 75% and 50%, and overall survival was 83% and 69%, respectively. Median overall survival was not reached and median progression-free survival was 23 months. Lenalidomide is well tolerated after allogeneic stem cell transplantation; the combination with DLI did not cause a higher risk of graft-vs-host disease; an immunological synergistic effect was probably present with this strategy. This combination should be evaluated further in a larger cohort of patients.

Dose-Adjusted EPOCH-Rituximab Combined With Fludarabine Provides an Effective Bridge to Reduced-Intensity Allogeneic Hematopoietic Stem-Cell Transplantation in Patients With Lymphoid Malignancies

There is currently no standard chemotherapy regimen for patients with lymphoid malignancies being considered for reduced-intensity conditioning allogeneic hematopoietic stem-cell transplantation (RIC-alloHSCT). The ideal regimen would provide disease control and result in lymphocyte depletion to facilitate engraftment. To this end, we developed a novel regimen by adding fludarabine to dose-adjusted continuous-infusion etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin plus with or without rituximab (DA-EPOCH-F/R). One hundred forty-seven patients with lymphoid malignancy (median age, 50 years) who had heavily pretreated (median prior regimens, three) and chemo-refractory (47%) disease were treated with DA-EPOCH-F/R before RIC-alloHSCT. Patients received one to three consecutive cycles until achieving lymphocyte depletion (CD4(+) count < 200/μL) or progressive disease. Overall response rate was 41%; 39% of patients had stable disease. Toxicity included grade 4 neutropenia in 65% and thrombocytopenia in 25% of patients. DA-EPOCH-F/R resulted in lymphocyte depletion (P < .001), which was inversely associated with serum interleukin (IL) 7 and IL-15 levels. Of 147 patients, 143 patients proceeded to RIC-alloHSCT. Patients with lower CD3(+) (P < .001), CD4(+) (P < .001), and CD8(+) (P < .001) T-cell counts after DA-EPOCH-F/R were more likely to achieve full donor lymphoid chimerism by day +14 after transplant. Relative to nonresponders to DA-EPOCH-F/R, patients with complete and partial response had increased event-free survival (77.4 v 4.8 months; P < .001) and overall survival (98.5 v 16.2 months; P < .001). DA-EPOCH-F/R safely provides tumor cytoreduction and lymphocyte depletion, thereby offering a bridge to RIC-alloHSCT in patients with aggressive lymphoid malignancies.

Decitabine in Combination with Donor Lymphocyte Infusion as Salvage Therapy for Relapsed AML Post-Allogeneic Stem Cell Transplantation

Prognosis is extremely poor for AML patients who relapse after allogeneic stem cell transplantation. Donor Lymphocyte infusions (DLI) can be used to salvage these patients with complete response rates reported to be 10-15% with an associated 40-60% chance of developing clinically significant GVHD. The mechanism by which DLI results in clinical responses is thought to be a T-cell mediated process. Data suggest that DLI normalizes the T-cell receptor repertoire and expands the anti-leukemic cell population. Hypomethylating agents, which appear to foster the graft versus leukemia phenomenon, were combined with DLI in attempt to enhance the graft versus leukemia effect. We report ten AML patients who received Decitabine +/- Etoposide with incremental DLI as salvage after relapse from allogeneic stem cell transplantation during the years 2007-2010. These patients were between the ages of 26- 73 years. Six patients had de novo AML. Three patients were transformed from MDS, and one from essential thrombocythemia. Eight patients had reduced intensity conditioning regimens. Two patients received a fully ablative preparative regimen. Average time to progression post transplant was 17 months. Patients received Decitabine at 20mg/m2 for 5 - 10 days, some in combination with Etoposide for 3-5days for disease control. Patients received 1-4 courses of treatment approximately every 28 days with DLI between days 14-21. Two patients who progressed while receiving Decitabine/Etoposide received Clofarabine at 20-52mg/m2 for 5 days, with subsequent DLI. Cell dose ranged from 1.27 to 31.7 CD3+ cells / kg. Two patients received a mobilized DLI. Six out of ten patients regained full chimerism. One patient who relapsed with extra medullary disease never lost his graft. Seven out of ten patients achieved a complete remission. Only one patient developed Grade 2 GVHD of the skin. Overall survival in these patients after relapse from allogeneic transplantation was 10.8 months. The combination of Decitabine and DLI is a well tolerated outpatient therapeutic option for patients with relapsed AML post allogeneic stem cell transplantation. The majority of patients regained full chimerism and achieved complete remission with little or no GVHD.

Acute Gvhd Is a Strong Predictor of Full Donor cd3+ t Cells Chimerism (TCC) After Reduced Intensity Conditioning (RIC) for Allogeneic Stem Cell Transplantation (Allo-SCT)

Abstract 4545The monitoring of chimerism is a standard procedure for assessing hematopoietic engraftment and achievement of full donor lymphoid chimerism after RIC based Allo-SCT. Post graft donor lymphocyte infusions are often decided on this evaluation. These studies have however a cost issue, all the more no consensus presently exists on when and how often to perform them.We retrospectively analysed the impact of acute GvHD in the prediction of allograft chimerism in our RIC program where TCC was serially assessed at 30, 60 and 90 days after Allo-SCT. We selected patients with hematologic malignancies (with the exclusion of myelofibrosis) transplanted between 2001 and 2010 after Fludarabine-Busulfan-ATG RIC from a HLA identical donor. 115 patients fulfilled all criteria including at least one T cells chimerism (TCC) determination between day 30 and 120. Allo-SCT was performed from familial donor in 92 patients (80%) and from MUD in 23 patients (20%). The conditioning regimen consisted of fludarabine (90 to 180 mg / m2), Busulfan (8 mg / kg orally or 6.4 mg / kg IV) and rabbit anti-thymocyte globulin (ATG) (2.5 or 5 mg /kg). As for chimerism study, recipient peripheral blood T lymphocytes were positively sorted by a mix of anti-CD4 and CD8 immunomagnetic beads (Dynal, Compiègne, France). T-cell purity was controlled by flow cytometry and was always > or =95%. Genomic DNA was amplified using fluorescent PCR primers for polymorphic variable number tandem repeats (VNTR) or short tandem repeats (STR). Mixed T-cell chimerism was defined as between 5 and 94% recipient cells, and full chimerism was defined as the presence of more than 95% donor cells.Full TCC was achieved in 94 patients (82%) at a median of 77 (30–120) days post transplant.Fifty eight patients (50.4%) developed acute GvHD. The cumulative incidence of Grade 2–4 GvHD in our population is 32% (95% CI 23–41).Overall the results showed that each of the 37 patients developing grade ≥ 2 AGVHD had a Full TCC prior day 120. On the other hand, all mixed chimerism were documented in patients not presenting Grade≥2 AGVHD (21 of the 78 patients (27%) without grade ≥ 2 AGVHD) (p=.002). No other parameter (ATG dose, Donor type…) achieved this level of individual prediction.These results, in a very homogenous population, are in line with the concept that full TCC is more likely to occur when patient develops significant aGVHD. Although they deserve further and deeper confirmation in different populations, they address the value of systematic routine chimerism surveillance (outside clinical studies) in patients presenting acute GvHD following RIC Allo-SCT. The modulation of TCC determination might represent an interesting cost and resources saving. Disclosures:No relevant conflicts of interest to declare.

The Effect of KIRs Expression Profile in Donor/Recipient Pairs in HLA-Identical Sibling Hematopoietic Stem Cell Transplantation,

Abstract 4017 Objective:We investigated the distribution characteristics of KIRs expression profile in donor/recipient pairs with acute leukemia (AL) receiving HLA-identical sibling hematopoietic stem cell transplantation (sib-HSCT). We further explored the effect of KIRs expression profile in donor/recipient pairs on clinical outcome, including dynamics of donor T cell and NK cell engraftment. Methods:The genotypes of donor/recipient KIRs were determinated by polymerase chain reaction- sequence specific primer (PCR-SSP) for 80 pairs of donor/recipient receiving HLA-identical sibling hematopoietic stem cell transplantation. The multiple short tandem repeat (STR) PCR was used to evaluate the status of engraftment of donor T cells and NK cells at +14 days, 21 days, 28 days, 60 days and 90 days after transplantation in 24 cases. Results: 1.In 80 pairs of donor/recipient: (i) the KIRs were completely identical in 57.5% of donor/recipient pairs; (ii) the donors' KIRs contained the recipients' in 13.75% pairs; (iii) the recipients' KIRs contained the donors' in 17.5% of pairs; (iv) the KIRs were completely different in 11.25% pairs. The graft versus host (GVH) direction KIR-matched group was 75%. The percentage of group donor B/X and group donor A/A was 50%, respectively. 2. Comparing the patients from GVH direction KIR-matched and mismatched group, the incidence of acute (a) GVHD was 60% and 30%, respectively (P =0.0222), and 2-year OS was 62.96% and 94.12%, respectively (P =0.0492). Particularly, grade III-IV aGVHD rate of KIR-matched group was higher than that of non-KIR matched group(15% vs 0%). 3. Donor B/X group had a higher 2-year OS and 2-year relapse-free survival (RFS) compared with donor A/A group (89.23% vs 49.57%, P =0.0159, and 90% vs 59.71%, P =0.0239, respectively). Patients with three or less aKIRs had a lower 2-year OS (58.9% vs 92.44%, P =0.0338) and a lower RFS (65.14% vs 92.59%, P =0.0398), compared with patients with more aKIR. 4. Sequential monitoring of chimerism status of donor NK-cells in 24 cases revealed that on day+14, the percentage of full donor NK cells chimerism was higher in non-KIR matched patients than that of KIR matched patients (85.7% vs 52.9%, P =0.0456). Conclusions:Donor KIR genotype appears to have a direct impact on aGVHD, OS and RFS. Therefore, donor KIR genotype should be evaluated as an outcome predictor of the HLA-identical sib-HSCT. Disclosures:No relevant conflicts of interest to declare.

Co-Infusion of Allogeneic Cord Blood with Haploidentical CD34+ Cells Improved Transplant Outcome for Patients with Severe Aplastic Anemia Undergoing Cord Blood Transplantation

Abstract 654Unrelated cord blood (UCB) transplantation is a useful alternative for patients with hematological malignancies or non-malignant hematological disorders lacking an HLA matched donor. However, outcomes for patients with severe aplastic anemia (SAA) undergoing either a single or dual UCB transplant have been disappointing. A recent EBMT/ Eurocord study reported engraftment and 3 year survival rates of only 51% and 38% respectively (Perrault de Latour, Biol Blood Marrow Transplant 2011). We investigated whether co-infusion of a single UCB unit with CD34+ selected cells from a haploidentical relative following a highly immunosuppressive conditioning regimen could improve transplant outcome for patients with SAA refractory to immunosuppressive therapy that lack an HLA matched donor. Subjects with SAA and life-threatening neutropenia (ANC <500) refractory to 2 or more immunosuppressive agents were eligible for enrollment if they lacked an HLA matched related or unrelated donor. Conditioning consisted of cyclophosphamide (120 mg/kg), fludarabine (125 mg/m2), equine ATG (160 mg/kg) and one dose of 200 cGy of total body irradiation. Patients received a G-CSF mobilized, T-cell depleted CD34+ selected stem cell product prepared from a haplo-identical donor using the Miltenyi CliniMacs system combined with a single ≥ 4/6 HLA antigen matched UCB unit. Eight patients with treatment refractory SAA (median age 18 years; range 9–20), including 1 patient with SAA evolved to MDS, have been transplanted. All patients were platelet and RBC transfusion-dependent with severe neutropenia (median ANC 60 neutrophils/ul; range 0–260). Patients were at high risk for graft rejection, being heavily transfused, having failed a median 3 (range 2–4) immunosuppressive regimens, and 4 (50%) were HLA alloimmunized. Six patients received a single 4/6 HLA matched UCB unit and 2 received a 5/6 HLA-matched unit. Transplanted allografts contained a median 2.9 × 107 CB TNCs/kg (range 2.6–7.3), 1.5 × 105 CB CD34+cells /kg (range 0.4–3.2), and 3.3 ×106 CD34+cells/kg (range 2.6–3.6) from the haploidentical relative. All patients achieved the primary study endpoint of donor engraftment with an ANC of >500 by day 42, 7 of 8 achieving a UCB-derived ANC >500 cells/μl. The median time to neutrophil recovery was 10 days (range 10–18 days). One patient failed to engraft with the cord unit, but has had sustained engraftment from the haploidentical donor, and is transfusion independent with a normal neutrophil count >25 months post transplant. Acute GVHD grade II developed in 2 patients and one developed limited chronic GVHD. Early T-cell engraftment was predominantly UCB in 7 cases; on day 21, T-cell chimerism was a median 100% cord in origin (range 0–100%). In contrast, myeloid chimerism at engraftment was predominantly haplo-donor in origin and showed 3 phases of engraftment: 1) early myeloid engraftment from the haplo-CD34+ cell donor 2) delayed myeloid engraftment from the cord unit resulting in dual myeloid chimerism and 3) disappearance of the haplo-donor cells with transition towards full cord donor myeloid chimerism (see figure).Mixed lymphocyte reactivity assays performed on post transplant PBMCs showed increasing alloreactivity of cord blood T-cells against the haploidentical donor during the period when myeloid chimerism transitioned towards cord, indicating that the disappearance of haplo-donor myeloid cells occurred as a consequence of rejection by engrafting cord blood T-cells. At a median follow-up of 9 months (range 75 days to 3 years), 7 patients survive, and all are transfusion–independent. One patient died 14 months after transplantation from complications related to CMV pneumonitis.In conclusion, transplantation of haploidentical CD34+ cells can shorten the time to neutrophil recovery in SAA pts undergoing a single UCB transplant. Furthermore, durable full engraftment from donor haploidentical CD34+ cells can occur in the context of cord graft failure. These data suggest co-infusion of allogeneic cord blood with haploidentical CD34+ cells can improve the outcome of UCB transplantation for SAA. [Display omitted] Disclosures:Wilder:NCI: Funded in part by NCI contract No. HHSN261200800001E.

Kinetics of Engraftment and Predictors of Outcome in Patients with Advanced Chronic Lymphocytic Leukaemia (CLL) After Treatment with Low Dose Total Body Irradiation (TBI) Followed by related or unrelated hematopoietic Stem Cell Transplantation

Abstract 4532 Introduction:In an unicenter analysis we investigated the impact of allogeneic hematopoietic cell transplantation (RIC-HCT) after reduced intensity conditioning on the kinetics of engraftment and predictor of outcome in 50 patients with advanced CLL. Patient and Methods:Patients in advanced stage CLL (n=50) received Fludarabine 30mg/m2 on day -4 to -2 and 2Gy TBI on day 0 followed by cyclosporine and MMF from unrelated (n=40) or related (n=10) donors between June 1999 and March 2010 at the University of Leipzig. The majority of patients (n= 35) were male, had Binet C (n=37, 74%) at RIC-HCT. The median age was 58 (range 44–69) years. Of 48 patients for whom cytogenetic analyses were available, 22 (46%) had unfavourable cytogenetics including del 17 or del 11q. Three (6%) patients had CR, 27 (54%) PR, 7 (14%) progressive disease, 12 (24%) stable disease and one (5%) relapse at RIC-HCT. Resistance to first line therapy was present in 25 (50%) patients, whereas 12 (24%) were resistant to Fludarabine. Richter's transformation was found in six patients (12%). Chimerism was detected in bone marrow and peripheral blood on T-lymphocytes and B-lymphocyte subpopulation after sorting at monthly intervals in the posttransplant period and than in 6 months interval. Results:Hematological toxicities after RIC-HCT were moderate. The majority of patients (96%) engrafted with neutrophiles >500/μ L median at day 22 after HCT. Six (12%) and 15 (30%) patients maintained absolute neutrophil counts (ANC) >0,5 × 109/L and platelet counts >50 × 109/L, respectively. T-cell donor chimerism increased to >95% at day 56 and B-cell donor chimerism to 94% at day +360, respectively. B-CLL cells disappeared completely on day +360 (median 0%). Overall survival (OS) at 4 years was 51%, Non relapse related mortality (NRM) 30%, Progression-free survival 33% and progression/relapse 37%. The most common causes of NRM were GvHD (n= 7; 14%) and sepsis (n=3, 6%). Factors significantly associated with increased risk of relapse/progression were intermediate/advanced disease vs. CR/PR1 (p=0.022) and lymphocytes ≥ 5 × 109/L vs. < 5 × 109/L (18% vs. 58%, p= 0.00) at 12 months in univariate analysis. Conclusion:Full donor T-cell chimerism was reached early after HCT, while B-cell reconstitution was observed only 1.5 years after RIC-HCT despite the absence of evident disease by 360 days after RIC-HCT. Best predictor for Progression-free survival (PFS) was CR or PR1. Disclosures:Pönisch:Mundipharma: Honoraria, Research Funding.