Chimerism Analysis Research Articles

Chimerism Analysis of Cell-Free DNA in Patients Treated with Hematopoietic Stem Cell Transplantation May Predict Early Relapse in Patients with Hematologic Malignancies.

Background. We studied DNA chimerism in cell-free DNA (cfDNA) in patients treated with HSCT. Methods. Chimerism analysis was performed on CD3+ cells, polymorphonuclear (PMN) cells, and cfDNA using 16 small tandem repeat loci. The resulting labeled PCR-products were size-fractionated and quantified. Results. Analyzing samples from 191 patients treated with HSCT for nonneoplastic hematologic disorders demonstrated that the cfDNA chimerism is comparable to that seen in PMN cells. Analyzing leukemia patients (N = 126) showed that, of 84 patients with 100% donor DNA in PMN, 16 (19%) had evidence of clinical relapse and >10% recipient DNA in the plasma. Additional 16 patients of the 84 (19%) showed >10% recipient DNA in plasma, but without evidence of relapse. Eight patients had mixed chimerism in granulocytes, lymphocytes, and plasma, but three of these patients had >10% recipient DNA in plasma compared to PMN cells and these three patients had clinical evidence of relapse. The remaining 34 patients showed 100% donor DNA in both PMN and lymphocytes, but cfDNA showed various levels of chimerism. Of these patients 14 (41%) showed laboratory or clinical evidence of relapse and all had >10% recipient DNA in cfDNA. Conclusion. Monitoring patients after HSCT using cfDNA might be more reliable than cellular DNA in predicting early relapse.

The Development of Methods for High Purity Hematopoietic Lineage Cell Isolation and Lineage Chimerism Testing

Chimerism analysis has become an important tool for assessing the origin of hematopoietic cells after allogeneic donor stem cell transplantations. Particularly, lineage chimerism analysis provides crucial information regarding treatment progress. Donor chimerism status for certain lineages, such as T, NK and myeloid, during early stages following post-transplant is associated with long term engraftment success and the relapse risk. However, the accurate lineage chimerism result is highly contingent on obtaining lineage cell preparation purity. There are several challenges in the field relating to the isolation of high purity cell lineages, chiefly among which is extremely low cell counts during the early stages post-transplant. Our laboratory has developed a method based on a) sequential isolation of hematopoietic lineages; b) purity assessment by flow cytometry; and c) a correction algorithm substandard purity of isolated cells. First, myeloid, B, T, and NK cells are sequentially isolated by magnetic separation using lineage specific markers. Thus each lineage isolated is largely excluded from the sample as a potential contaminant in each subsequent step. A purity with >90% can be routinely achieved when isolating lineages accounting for as little as 0.5 % of total leukocytes. Second, the purity of each population is verified by Flow Cytometry using a 6-color antibody panel staining for T, B, NK, myeloid, and total leukocytes. This enables not only the determination of cell purity of each isolated lineage cells, but also the identification and quantification of any contaminating cells in each isolated lineage. Lastly, an algorithm was derived based on the assumption that total chimerism consists of the four main lineages, T, B, NK, and myeloid. This mathematical correction is reflexively applied when isolated lineage cells are of <90% purity, often occurred as the isolated lineage accounting for < 0.5% of total leukocytes in the sample. In summary, our methods enable generation of high purity of isolated lineage cells that lead to accurate and reliable single lineage chimerism results.

Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1

ABSTRACTMAP1LC3/LC3 (a mammalian ortholog family of yeast Atg8) is a ubiquitin-like protein that is essential for autophagosome formation. LC3 is conjugated to phosphatidylethanolamine on phagophores and ends up distributed both inside and outside the autophagosome membrane. One of the well-known functions of LC3 is as a binding partner for receptor proteins, which target polyubiquitinated organelles and proteins to the phagophore through direct interaction with LC3 in selective autophagy, and their LC3-binding ability is essential for degradation of the polyubiquitinated substances. Although a number of LC3-binding proteins have been identified, it is unknown whether they are substrates of autophagy or how their interaction with LC3 is regulated. We previously showed that one LC3-binding protein, TBC1D25/OATL1, plays an inhibitory role in the maturation step of autophagosomes and that this function depends on its binding to LC3. Interestingly, TBC1D25 seems not to be a substrate of autophagy, despite being present on the phagophore. In this study we investigated the molecular basis for the escape of TBC1D25 from autophagic degradation by performing a chimeric analysis between TBC1D25 and SQSTM1/p62 (sequestosome 1), and the results showed that mutant TBC1D25 with an intact LC3-binding site can become an autophagic substrate when TBC1D25 is forcibly oligomerized. In addition, an ultrastructural analysis showed that TBC1D25 is mainly localized outside autophagosomes, whereas an oligomerized TBC1D25 mutant rather uniformly resides both inside and outside the autophagosomes. Our findings indicate that oligomerization is a key factor in the degradation of LC3-binding proteins and suggest that lack of oligomerization ability of TBC1D25 results in its asymmetric localization at the outer autophagosome membrane.

Phase II Study of Haploidentical Natural Killer Cell Infusion for Treatment of Relapsed or Persistent Myeloid Malignancies Following Allogeneic Hematopoietic Cell Transplantation

We conducted a phase 2 study to determine the efficacy of HLA-haploidentical related donor natural killer (NK) cells after cyclophosphamide-based lymphodepletion in patients with relapsed or progressive acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) following allogeneic hematopoietic cell transplantation (HCT). Eight patients (2 with MDS and 6 with AML) were treated with cyclophosphamide 50 mg/kg on day −3 and day −2 before infusion of NK cells isolated from a haploidentical related donor. One patient also received fludarabine 25 mg/m2/day for 4 days. Six doses of 1 million units of interleukin-2 (IL-2) were administered on alternating days beginning on day −1. The median number of NK cells infused was 10.6 × 106/kg (range, 4.3 to 22.4 × 106/kg), and the median number of CD3 cells infused was 2.1 × 103/kg (range, 1.9 to 40 × 103/kg). NK infusions were well tolerated, with a median time to neutrophil recovery of 19 days (range, 7 days to not achieved) and no incidence of graft-versus-host disease after NK infusion. One patient with AML and 1 patient with MDS achieved a complete response, but relapsed at 1.7 and 1.8 months, respectively. One patient with MDS experienced resolution of dysplastic features but persistence of clonal karyotype abnormalities; this patient was stable at 65 months after NK cell therapy. The median duration of survival was 12.9 months (range, 0.8 to 65.3 months). Chimerism analysis of CD3−/CD56+ peripheral blood cells did not detect any circulating haploidentical NK cells after infusion. NK phenotyping was performed in 7 patients during and after IL-2 infusion. We found a slight trend toward greater expression of KIR2DL2/2DL3/2DS2 (5% versus 28%; P = .03) at 14 days in patients who survived longer than 6 months from NK cell infusion (n = 4) compared with those who died within 6 months of NK cell therapy (n = 3). In summary, our data support the safety of haploidentical NK cell infusion after allogeneic HCT.

Graft-versus-host Disease After Intestinal or Multivisceral Transplantation: A Scandinavian Single-center Experience

BackgroundGraft-versus-host disease (GVHD) that develops after intestinal or multivisceral transplantation is difficult to diagnose and is associated with high morbidity and mortality. Material and MethodsThe objectives of this study were to investigate the incidence, clinical picture, risk factors, and outcome of GVHD in a Scandinavian cohort of patients who underwent intestinal or multivisceral transplantation during a period of 16 years (1998–2014). All transplanted patients (n = 26) were retrospectively analyzed with respect to donor- and recipient-derived risk factors. The diagnosis of GVHD was based on clinical signs, chimerism analyses of leukocytes, and histopathologic findings in biopsy specimens. ResultsFive of 26 patients (19%) were diagnosed with GVHD, of which three had skin GVHD, one had skin and bone marrow GVHD, and one had passenger leukocyte syndrome. Only multivisceral-transplanted patients developed GVHD. Risk factors for development of GVHD were an underlying tumor diagnosis and neoadjuvant chemo- or brachytherapy administered before intestinal transplantation. All patients were given high-dose corticosteroids as first line treatment for their GVHD, and all survived their episodes of GVHD. ConclusionsThe risk of GVHD appears to be increased in recipients of multivisceral transplantations who received chemotherapy due to an underlying malignancy. The reasons may be the large amount of lymphoid tissue in these types of grafts, and the cytotoxic effects of the malignancy and chemotherapy on healthy recipient tissues. These patients should be monitored closely for the development of GVHD.

Allogeneic SCT in Multiple Myeloma: Limited Predictive Value of Chimerism Analysis for Heralding Relapse/Progression Due to Extramedullary Disease

Background Allogeneic stem cell transplantion (allo SCT) for multiple myeloma (MM) takes advantage of a tumor cell-free graft along with potential immunosurveillance of residual malignant plasma cells. However, multiple factors such as treatment-related mortality (TRM) and morbidity and post-allo disease progression question the more widespread use of allo SCT. The induction of an early complete donor chimerism (DC) is thought to be associated with a low incidence of relapse and improved survival. Interestingly, data on chimerism analysis are relatively scarce for allo SCT in MM. Therefore, we retrospectively evaluated longitudinal DC results by short tandem repeat (STR)-based techniques from a single lab. Data were correlated with a set of baseline and outcome variables. In addition, disease- and treatment related factors like extramedullary disease (EMD), acute and chronic graft-versus-host disease (GvHD) and post-transplant consolidation were analyzed for impact on outcome.Patients and methods We identified 118 MM patients (pts) receiving non-myeloablative allo SCT at two German centers between 01/2006 and 12/2014. 90% of pts underwent allo SCT at relapse. Median follow-up was 42 months (mos), median overall survival (OS) 25 mos (range, 1-106) and TRM was 15%. Two-thirds of pts had received treosulfan/fludarabine as conditioning regimen. Median interval between MM diagnosis and allo SCT was 40.5 mos (range, 7-175) and median number of prior treatment lines was 3 (range, 1-10). 24 pts received allografts from matched related donors, 61 from matched-unrelated donors, and 33 pts from mismatched donors. Chimerism analysis was performed by STR-PCR on whole blood and T cell subsets using a validated multiplex STR-assay with a documented sensitivity of 1%. Response assessment was performed according the international myeloma working group guidelines.Results 1398chimerism samples (mean: 12/patient, 119 marrow, 1279 blood)) were analyzed. Median interval from allo SCT to first assessment was 15 (range, 3-62) days. Median duration of chimerism sampling was 24 mos. No graft rejections occurred. Full DC (> 99%) was achieved in 117/118 pts. Pts who lost full DC had a significantly shorter progression free survival (PFS) than those with sustained DC (HR 0.2; p<.0001). OS, however, was not different. In patients who developed disease progression, loss of DC versus sustained DC was not predictive for outcome. In spite of relapse, a full DC was found in peripheral blood in 82% and in 45% in bone marrow samples of pts, respectively. With respect to clinical predictors of outcome, the occurrence of acute GvHD had no impact on PFS nor OS. In contrast, induction of chronic GvHD was associated with prolonged PFS (HR 0.50; p=.0088) and OS (HR 0.39; p<.001). Despite the induction of full DC in the blood, 13/118 (11%) of pts not in complete response received preemptive donor lymphocyte infusions (DLI). DLI without any concomitant signs of graft rejection or disease progression was associated with favourable OS (HR 0.43; p=.0067). When analysing relapse patterns after allo SCT, a 35% rate of EMD was observed. Interestingly, OS was not different when compared to intramedullary relapses. However, when allo SCT was performed as part of a consolidation therapy for prior EMD or plasma cell leukemia, OS was significantly shorter than compared to patients without EMD history (median OS 6 vs. 41 mos, p=.0035) Nevertheless, 25% of these EMD patients showed long-lasting remissions beyond 4 years. High risk cytogenetics (del17p, t(4;14) and +1q21), CMV reactivation or use of ATG had no impact on outcome.Conclusions To the best of our knowledge, this is the largest study of serial DC analysis in a cohort of clinically well characterized MM pts. The robust engraftment in almost all patients as well as the lack of graft rejections indicate the severe immunosuppression in MM due to pre-treatment and underlying disease. On the other hand, the most important predictors for PFS and OS was the occurrence of cGvHD. Preemptive DLI may further improve tumour control. The frequent spread of myeloma to extramedullary sites can be interpreted as an escape to more immuno-privileged regions as compared to the bone marrow. This pattern clearly limits the potential of prolonged serial DC analysis as the only tool for relapse detection. Our observations underscore the need to prospectively evaluate complementary strategies in this setting. DisclosuresEinsele:Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Thiede:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AgenDix GmBH: Equity Ownership. Knop:Celgene Corporation: Consultancy.

Allogeneic Stem Cell Transplantation Using In Vivo T-Cell Depletion in Myeloid Disorders

Introduction:Alemtuzumab is used for in vivo T-cell depletion to reduce graft versus host disease in allogeneic Stem Cell Transplantation (SCT). Profound lymhotoxicity of this monocloncal antibody can potentially increase morbidity and mortality in SCT due to excessive viral infections and increased risk of graft rejection. We retrospectively analysed outcome of patients with myeloid disorders (acute myeloid leukaemia and myelodysplasia) who received in vivo T-cell depletion using Alemtuzumab based conditioning for allogeneic SCT over a period of 3 years in our centre.Methods:Patients were identified from department transplant data base. Data was collected for 73 consecutive patients over a period of three years using patient medical records, clinical work station and electronic patient records. The conditioning regimen included Fludarabine 30mg/m2 x5 (days -7 to -3), Alemtuzumab 10mgx5 (days -8 to -4) and Melphalan 140mg/m2 (day -2). Chimerism analysis was performed by polymerase chain reaction (PCR) to identify short tandem repeats within peripheral blood leucocytes and CD3 fraction. The quantification of donor chimerism was done by using gel photography system and LabWroks software. Viral testing was performed by PCR analysis.Results:Median age for the patients was 59 years (range 41-71). Median duration of follow up was 19 months (4-49 months). The majority of patients (67%) received SCT from a voluntary unrelated donor. Sixty four patients (88%) had AML whilst 9 had myelodysplasia. Sixty five (89%) patients were in morphological complete remission (<5% blasts) at the time of SCT. Median HCT-Comorbidity index was 2 (range 1-5). Median time to neutrophil engraftment was 13 days (range 9-23) and platelet engraftment 14 days (median 8-48). During the course of transplantation, 50 patients were treated for neutropenic fever with broad spectrum antibiotics and 16 for presumed fungal infection. Forty three patients (59%) reactivated cytomegalovirus (CMV), 10 (13%) had Ebstein Barr Virus (EBV) viremia and 4 (5%) were found to have adenovirus on peripheral blood PCR analysis after SCT. Denovo acute graft versus host disease (grade 2-4) required treatment in 12 patients (16%) whilst chronic GVHD was seen in 14% (n=10). 20 patients required donor lymphocyte infusion due to mixed chimerism after SCT. Total incidence of acute GVHD (grade 2-4) was 23% (n=17) and that of chronic GVHD was 15%. Median donor chimerism in peripheral blood leucocyte fraction was 100% at day 30, 60, 90 and 180 after SCT. Chimerism analysis on CD3 compartment showed 100%, 96%, 95.5% and 95% donor fraction respectively at these time points. Predicted overall survival at 2 years was 53% and Event Free Survival 49%. Non relapsed mortality in this cohort was 18% at one year. One patient had primary graft rejection with no secondary graft rejections. Overall survival was not statistically different between those who were treated for viremia (CMV, EBV, adenovirus) when compared to those who did not have viremia (p=0.31). No deaths were attributed to either CMV or EBV.Conclusion:Our retrospective data show that Alemtuzumab based conditioning regimen in myeloid disorders have low incidence of GVHD, very low risk of graft rejection and comparable overall survival to those conditioning regimens which utilize in vivo T-cell depletion strategies other than Alemtuzumab. Moreover, a high incidence of viremia, in our cohort, did not translate into worse overall survival. DisclosuresSaif:Novartis: Honoraria; Alexion: Honoraria. Dignan:Jazz pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Therakos: Honoraria, Speakers Bureau. Tholouli:Jazz Pharmaceuticals: Honoraria, Speakers Bureau; MSD: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau.

Donor Lymphocyte Infusion in Patients with Thalassemia Major Who Have Mixed Chimerism Following Allogeneic Stem Cell Transplant

▪Allogeneic stem cell transplantation (HSCT) is the only curative option for patients with thalassemia major (BTM). HSCT in BTM is associated with increased risk of graft rejection. Some patients do not achieve full donor chimerism after transplant despite hematopoietic engraftment. Early post transplant mixed chimerism (MC) is a known predictor of secondary graft rejection. Data is limited on the role of donor lymphocyte infusion (DLI) to prevent secondary graft rejection following detection of early MC in BTM. We report our experience with DLI in BTM patients who had developed progressive worsening early MC after HSCT.All patients with BTM who underwent HSCT at our centre between September 1994 and December 2013 were included in the analysis. During this period, 406 patients underwent HSCT for BTM. Median age was 8 years (range: 1 - 25) and 247 were males (60.8 %). Conditioning regimen was busulphan based in 274 (67.5%) or treosulfan based in 132 (32.5%). The graft source was BM in 305 (75.1%) and PBSC in 101 (24.9%) etc. Median CD34 stem cell dose was 9.24x106/kg (range: 2.1- 33). GVHD prophylaxis consisted of CSA and short course MTX.Patients who engrafted underwent chimerism analysis of peripheral blood at day 28 and between days 60-90. Subsequently chimerism analysis was done at the discretion of the treating physician. Chimerism analysis was done by qualitative and quantitative methods up to 2007 and by quantitative method from 2007.MC was defined by >5% recipient cells at any time point post transplant. The severity of MC was determined as per previously published criteria (level I= < 10%, level II = 10-25 %, level III = >25 %). In patients who developed MC, immunosuppression was tapered and stopped and chimerism monitored. Cyclosporine was tapered by approximately 30% every 2 weeks until there was stable MC or complete donor chimerism. If progressive loss of donor chimerism leading to level II or III MC occured on two consecutive occasions with a concurrent drop in hemoglobin despite stopping immunosuppression and in the absence of GVHD, DLI was given. Patients should have had at least 10% donor chimerism in order to have a DLI.There were 109 (41.6 %, out of 262 patients in whom chimerism was done) patients who developed MC. Of these, 35 (32.1%) developed progressively worsening MC and drop in hemoglobin despite tapering of immunosuppression (91% of these were within day 60). Of these 35 patients, 23 (65.7%) received DLI while 12 developed anemia requiring transfusion (4continued to be on blood transfusion and 8 underwent a second transplant).Among 23 patients who received DLI, 9 (39.1%) showed response (rise in Hb with CC or stable MC) while 14 (60.9%) failed to show any response. The median time to show response was 28 (8-192) days and the median time to peak response was 44 (23-442) days. The 14 patients who did not show any response developed anemia requiring transfusion (11 continued to be on blood transfusion while 3 underwent a second transplant). While 80% of those with level II chimerism responded to DLI, only 31.2% of those with level III chimerism showed a response (summarized in Table 1).DLI was well tolerated by the majority of the patients. However, 7 (30.4%) patients developed cytopenia. While 6 of these had level III chimerism at the time of DLI, 1 had level II chimerism. Two patients developed Grade IV cytopenia. Grade 2 acute GVHD developed in 3 (13%; liver=2, gut=1) which responded to immunosuppressive treatment. Chronic GVHD developed in 2 (8.7%: extensive=1). There was no mortality related to DLI.DLI helped close to 40% patients achieve either complete donor chimerism or stable MC who otherwise might have required a second stem cell transplant with minimal risk. There was a trend to suggest that early administration of DLI at level II MC was superior to DLI administered at level III.Table 1Factors affecting the response to DLIn(%)Effect of DLIResponse(CC+Stable MC)No responsepChimerism levelI 000II 54(80%)1III 165(31.2%)110.07MC 202Lucarelli classI 11(100%)0II 83(37.5%)5III 145(35.7% )90.4ConditioningBu-Cy-ATG/ALG 124(33.3%)8Thio-Treo-Flu 115(45.5%)60.7GraftBM 175(29.4%)12PB 64(66.7%)20.1Time of 1st DLI<180 days 187(38.9%)11>180 days 52(40%)31.0Total239(39.1%)14 DisclosuresNo relevant conflicts of interest to declare.

Real-Time PCR Analysis of Chimerism in T Cell Subsets as an Early Predictor of Graft-Versus-Host Disease Following Allogeneic Stem Cell Transplantation.

Graft-versus-host-disease (GvHD) is the major cause of morbidity and mortality after stem cell transplantation. The development of early prediction methods is therefore of importance. Our aim was to analyze the usefulness of early donor chimerism monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in T cells and in CD4+ and CD8+ (lineage chimerism) for GvHD prediction. Chimerism was analyzed in 76 consecutive adult patients using RQ-PCR TaqMan technology on DNA extracted from Pan T, CD4+, and CD8+ cell subsets on Day 5, 10, 15 and 30 after allo-HSCT. The threshold of chimerism predictive for GvHD was the same for all tested cell subsets. In acute myeloid leukemia (AML) patients treated with myeloablative conditioning (MAC), the threshold predictive for acute graft versus host disease was 95% and 99% for Day 10 and Day 15, respectively. In patients treated with reduced intensity conditioning (RIC), the threshold predictive for chronic graft versus host disease was 98% on Day 10. The differences were statistically significant. Chimerism analysis in T cell subsets by RQ-PCR on Day 10 and Day 15 after transplantation is useful for prediction of aGvHD (AML patients after MAC) and cGvHD (patients after RIC). However, there was no difference in the results between chimerism in the T cell subsets. Our RQ-PCR protocol was highly sensitive and proved effective for analysis of lineage chimerism.

Mixed Chimerism at Three Months after Allogeneic Hematopoietic Stem Cell Transplantation Is a Significant Predictor of Disease Relapse in High-Risk Acute Myeloid Leukemia Patients in First Complete Remission

Background:Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative strategy for high-risk acute myeloid leukemia (AML) patients. However, the risk for disease recurrence following allo-HSCT remains significant and associated with poor outcomes. The ability to predict relapse before detectable morphologic recurrence may allow for preemptive interventions, such as immune modulation, donor lymphocyte infusion (DLI), or initiation of hypomethylating agents, to potentially augment graft-versus-leukemia effect. Post-transplantation peripheral blood chimerism analysis, represents a potential tool to predict disease recurrence, although a validated consensus on the use of this technique in the post-allo-HSCT follow-up has not been established yet and its precise role in this setting remains unclear. The aim of our study is to evaluate the impact of chimerism evaluation 3 months after allo-HSCT in AML patients who were in first complete remission (CR1) before transplantation and remained in clinical and morphological CR at day 90 post-transplantation, on overall survival (OS) and relapse incidence.Patients and methods:We evaluated 194 AML patients who received allo-HSCT at our center between January 2006 and December 2014 and for whom chimerism follow-up has been performed at 3 months. There were 103 (53%) males and 91 (47%) females with a median age of 43 years (range: 18-67). Patients were classified according to the European LeukemiaNet classification for cytogenetic and molecular biology markers (Dohner et al. Blood 2010), accordingly, 64% patients were unfavorable and 36% were in intermediate II risk group. At allo-HSCT, all patients were in CR1; 136 (70%) received a full intensity conditioning (MAC) and 58 (30%) received a reduced intensity conditioning (RIC). HSC donors were 72 (37%) HLA identical siblings (44 BM and 36 PBSC), 54 (28%) 10/10 HLA matched unrelated donors (35 BM and 19 PBSC), 33 (17%) 9/10 HLA mismatched unrelated donors (16 BM and 17 PBSC) and 35 (18%) double cord blood units (only 7 were 6/6 HLA matched). Chimerism analysis was performed on marrow and/or blood samples every month following allo-HSCT using polymerase chain reaction (PCR) based on informative polymorphic short tandem repeat, a mixed chimerism was defined by having 5% or more of recipient cells.Results:At day 90 after transplantation, all patients remained in clinical and cytological CR at time of chimerism evaluation, 155 (80%) had a full donor chimersim (FDC) and 39 (20%) had a mixed chimerism (MC) ranging from 65% to 95% of donor cells. Among patients with MC, 9 (23%) received increasing doses of DLI (5 of them reached FDC at 6 months), while 20 (51%) could not receive DLI (7 because transplanted from cord blood, and 13 because of the presence of GVHD), the rest of patients were left with a transient MC and regained FDC during follow-up.After a median follow-up of 34 months (range: 4-96) for the surviving patients, the median OS in patients with FDC was not reached with a 3 years probability of 62% (95% CI: 58-66), and for patients with MC, it was 18 months (12-24) with a 3 years probability of 32% (95% CI: 23-41), (p=0.01). Twenty-two patients in the MC group have progressed during the follow-up and 17 among them died from disease progression. The cumulative incidence of relapse at 3 years was 25% (95% CI: 21-29) for FDC patients and it was 70% (95% CI: 62-80) for MC patients, (p<0.001). The impact of mixed chimerism was still valid in multivariate analysis after including patient age, type of donor, HSC source and risk group, and was independent from the intensity of the conditioning regimen with a Hazard Ratio of 4.7, and a 95% CI: 2.6-8.4, p<0.001.Conclusion:We demonstrated that chimerism evaluation at day 90 after allo-HSCT is an independent predictor of disease relapse in patients who remain in CR at that time and significantly impacts on long term survival. The standardization of this evaluation may lead to the identification of patients with high-risk of relapse risk suggesting the need of early preemptive intervention. [Display omitted] DisclosuresNicolini:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ariad Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Low Expression of p53 Protein in Bone Marrow after Hematopoietic Stem Cell Transplantation Indicates Risk of Relapse in Pediatric Patients with Acute Lymphoblastic Leukemia

Advances in treatment over the last decades have resulted in almost 90% 5-year survival of children with acute lymphoblastic leukemia (ALL). Nonetheless, a minor group with unfavorable prognosis becomes candidates for hematopoietic stem cell transplantation (HSCT). After HSCT, blast count, minimal residual disease (MRD) and chimerism analysis are used as markers for successful engraftment and risk of relapse. However, additional markers are needed to complement the existing ones, as 30 % of ALL-patients still experience relapse after HSCT. Mutations in the tumor suppressor gene TP53 are detected in 2% at diagnosis of pediatric ALLs, but in 11-28 % after relapse. The aim of this study was to evaluate if alterations in the expressions of the TP53 encoded protein p53, analyzed by immunohistochemistry, could act as a prognostic marker in pediatric ALL patients post HSCT.Paraffin-embedded bone marrow samples were collected retrospectively from 49 children diagnosed with pre-B ALL and, treated with HSCT at Karolinska University Hospital between 1997-2010. Patients samples were requested from six different times during the course of the disease; time of diagnosis, before HSCT and at routine controls after HSCT at 0-3, >3-6, >6-12 and >12-24 months. For no patient data was available from all time periods. In total 176 samples were collected. A control group consisted of fifty-five children, where a bone marrow biopsy had been performed due to suspected bone marrow disorder, but a malignant blood disease had been excluded.Tissue micro array (TMA) prepared microscope slides were stained for the p53 protein using a monoclonal mouse anti-human antibody (clone DO-7, Leica), which detects both the wild-type and mutant forms of p53. Protein expression was examined by two independent examiners in a light microscope at high magnification (40X). Cells positive for the p53-protein identified by brown nuclear staining, were counted and calculated as a percentage of total cells per sample for each antibody respectively. Mean number of cells counted per sample was 695. Some samples were lost prior to final analysis due to either; sample core lost during TMA (n=12) or, sample had <100 cells which was minimum number of cells for inclusion in analysis (n=10). The final analysis included 154 bone marrow samples from 49 patients and 55 control samples.Data was analyzed in Statsoft Statistica 12.7. Nonlinear logistic regression was used to evaluate predictive value of the protein. Data was analyzed independently at each of the following times; diagnosis, time of HSCT and at 0-3, >3-6, >6-12 and >12-24 months post HSCT. A p value < 0.05 was considered significant.Interestingly, we found that a low percentage of cells positive for p53 protein expression in the bone marrow at >3-6 months after HSCT was associated with increased risk of relapse in pediatric ALL patients, odds ratio 0.82 (confidence interval 0.68-0.99) p= 0.041. Figure I shows the range of p53 expression in the two groups individually at each time.At >3-6 months after HSCT, the cut off value was calculated to 3.3% p53 positive cells, and was correct for 16 of 20 non-relapse cases and for 9 of 14 relapse cases (overall 73.5% correct). All relapses occurred between 6-50 months after HSCT (mean=20, median=14).The lower amount of p53 positive cells in the relapsed patients compared to the non-relapsed patients could possibly be due to an underlying mutation in TP53 that has altered the ability of producing a protein. This would be in similarity with the literature where it is suggested that TP53 mutations are more common at relapse in pediatric ALL patients.In summary, a lower expression of p53 protein in the bone marrow at >3-6 months after HSCT was significantly associated with increased risk of relapse. Evaluation of p53 protein expression by immunohistochemistry, in bone marrow from pediatric ALL-patients that undergo HSCT may be a potential additional marker for predicting relapse. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Novel Quantitative PCR Approach Targeting Insertion/Deletion Polymorphisms (Indel-PCR) for Chimerism Quantification: Finally High Sensitivity and Quantification Capacity Together

IntroductionPost-hematopoietic stem cell transplantation (SCT) chimerism monitoring is important to assess engraftment, anticipate relapse and provide information on the development of graft versus host disease, facilitating therapeutic intervention. The aim of this study was to test the technical efficacy and clinical utility of a novel quantitative PCR approach targeting insertion/deletion polymorphisms (indel-PCR).Materials (or patients) and methodsThis study included 157 samples (81 bone marrow, 60 peripheral blood (PB), 11 T-cells, 2 myeloid cells, 2 CD34-cells, 1 NK-cells purified using immunomagnetic technology) of 24 patients who underwent SCT for haematological malignancies. Additionally, 2 sets of 11 artificial mixtures were created using PB leukocytes of two healthy subjects (a male and a female) with known percentages of male leukocytes (putative recipient): 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0.01, 0.Chimerism analysis was performed by the gold-standard STR-PCR (AmpFlSTR SGM Plus®, Life Technologies, USA) and by indel-PCR (Mentype® DIPscreen, Mentype® DIPquant, Biotype, Germany). Concordance between both methods was calculated with SPSS using intraclass correlation coefficient.ResultsThe number of informative loci identified with indel-PCR (>3/patient) was higher than with STR-PCR for all patients.Concordance between both methods for the 157 patient samples and 11 artificial mixtures was "very good" (intraclass correlation coefficient=0.96). Of note, analysis of artificial mixtures provides evidence of significantly (≥2 logs) higher sensitivity by indel-PCR (0.01%) than by STR-PCR (1%, Table 1). Moreover, indel-PCR shows unprecedented quantification capacity (Table 1). Out of the 168 samples analyzed, 32 were positive and 15 negative by both methods, while 121 were positive only by indel-PCR (95% with <1% recipient).Clinical outcome and chimerism dynamics of 24 patients are described in figure 1, data of 4 of them were censored (engraftment failure, disease progression, insufficient number of samples). All the samples presented complete donor chimerism by STR-PCR. Of the 9 patients who showed stable or decreasing percentage of recipient cells, only one (11%) presented extramedulary relapse. Of the 11 patients who presented increasing percentage of recipient cells in one determination, relapse occurred in 5 patients (45%, 3/6 patients who did not have further determinations, 2/4 who showed stable or decreasing recipient cell percentage, and 0/1 patient who presented increasing recipient cells in subsequent determinations).ConclusionThis novel indel-PCR is a simple and accurate technique that, in comparison with the current gold standard STR-PCR, shows very good concordance and provides higher rates of informative loci per patient, as well as unprecedented combined sensitivity and quantification capacity. Such features allow minutely monitoring chimerism dynamics and might improve clinical management of transplanted patients, specially predicting relapse in those patients who do not have a molecular marker available for disease follow up. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Long-Term Overall and Event-Free Survival after RIC Allogeneic Transplantation in Acute Leukemias in a Single Centre - Impact of Age and Donor Type?

BACKGROUND: Between January 2007 and December 2014, we transplanted 143 patients (Pts) using RIC- conditioning allogeneic transplant. 71 were transplanted for acute leukemias (AML and ALL) and high risk myelodysplastic syndromes (MDS) not eligible for myeloablative transplantation. Clinicians are usually scared to transplant older patients with MUD because of the higher risk of Treatment-related mortality (TRM). We thus reviewed retrospectively the impact of donor / recipient ages and donor-choice (familial related donor (RD) versus matched unrelated donors (MUD)) on the post-transplant outcome of the pts.POPULATION AND METHODS: We retrospectively studied in our cohort of 71 leukemia pts evaluable after RIC transplant, the overall survival (OS) and event-free survival (EFS), according to recipient ages (≤ 54yo vs ≥55yo) and donor types (RD vs MUD).Conditioning consisted of combinations of Busulfan-Fludarabin-ATG or Endoxan-Fludarabine-ATG. Immunosuppression was a combination of cyclosporine (CSA)/mycofenolate mofetil (MMF), Tacrolimus (TAC)/ MMF or TAC/ Sirolimus. Chimerism analyses were performed on d30, d90 and d180. Both study-groups were well balanced concerning transplant conditioning regimens, (ATG or not) RD vs MUD donors, type of leukemia and status of the disease at transplant. Our local EBMT database was the source for this analysis.RESULTS: 34 de novo AML (20 ≤54 yr and 14 ≥55 yr), 30 secondary AML and MDS (15 ≤54 yr and 15 ≥55 yr) and 7 ALL (5 ≤54 yr and 2 ≥55 yr) were enrolled in our trial. 24 pts received HLA identical RD in the group ≤54yr and 14 pts in the group ≥55 yr. The others pts received MUD transplant from HLA 9 or 10/10 donors (16 in the group ≤ 54yr and 17 in the group ≥ 55yr). The median follow-up was 22 (3-93) months. Engraftment rate was 100% in the studied cohort. TRM before d100 was 18,3% (15% ≤54 yo and 22,6% ≥ 55yo). Grade II-IV Acute GVHD (aGVHD) at d90 (16,6%) and Chronic GVHD rates (24%) were similar in both age populations. 5 yrs OS was not different according to the age of the recipient (≤54 yr versus ≥55 yr)(p = 0.9). However, there is a clear, difference in 5years OS when stratifying the two age groups according to donor-types, especially for older patients (≥55 yr) with MUD transplant. (pts ≤ 54yr : OS: RD: 31,6%; and MUD: 38%; pts ≥ 55yr : OS: RD: 27,8% and MUD 44,2%). This trend in a better OS in older MUD pts could be explained partially by a lower relapse-rate in the group ≥ 55 yr with a MUD as shown by EFS/5yrs (pts ≤ 54 yr : RD: 32%; ≤ 54yr MUD: 19%; pts ≥ 55yr RD: 21% and ≥ 55yr MUD 38,5 %). As reported already in transplanted CLL patients, this suggests a greater GVHD/GVL-effect in the MUD transplantation. We then further investigated the age of the donors and its correlation with aGVHD and cGVHD. In the RD RIC transplant group, median age of the donors was 49 yrs (range 35-69 years) with 37.5% of aGVHD and 42% of cGVHD. In the MUD RIC transplant cohort, donor median age was 31 yrs (20-55 years) with 12% of aGVHD and 32% of cGVHD. We previously reported that the number of CD8+ T cells and naïve CD4+ T cells decreases with the age of the donor, also supporting a better control of the disease with younger donors. This combination of younger donor age, lower aGVHD and a similar rate of cGVHD clearly explains the better outcome in pts transplanted with MUD. Our data requires confirmation in a larger series of patients.CONCLUSION: our observations confirm that: 1) RIC transplantation is a curative approach in acute leukemia whatever the age when the pts are fit enough to tolerate the transplant. 2) despite a higher TRM compared to younger patients, older patients seem to particularly benefit from MUD in terms of OS. 3) an increased EFS is observed in older pts transplanted with MUD (also younger) donors more likely due to a greater GVHD/GvL effect in the MUD setting. DisclosuresNo relevant conflicts of interest to declare.

Functional and Molecular Consequences of the Dnmt3aR882H Mutation in Acute Myeloid Leukaemia

The de novo DNA Methyltransferase 3A (DNMT3A) gene is one of the most commonly mutated gene in acute myeloid leukaemia with a normal karyotype (AML-NK). Approximately two thirds of mutations are heterozygous and affect codon R882, located within the methytransferase domain. Also, we and others have shown that these mutations occur in a significant proportion of haematologically normal people and behave as leukaemia-initiating changes.DNMT3A mediates the transfer of a methyl group from s-adenosylmethionine (SAM) to the 5' carbon of cytosine residues at CpG dinucleotides. The DNMT3A protein domains bind to unmethylated H3K4, a repressive histone mark, as well as the H3K36Me3 mark associated with active transcription. DNA methylation is linked to histone modifications and DNMT3A binds to class I and II histone deacetylases (HDAC) and histone methyltransferases (HMT), such as SETDB1 and G9a, as well components of Polycomb repressive complex 2 (PRC2).DNMT3AR882H has been reported to exert a dominant-negative effect on wild type (WT) DNMT3A, with a significant reduction of its enzymatic activity. In AML samples, the R882 mutation correlates with global hypomethylation, particularly at CpG islands, shores and promoters, although some promoter hypermethylation has also been reported. However, the molecular mechanism, through which DNMT3AR882H alters methylation to drives leukaemia, remains unknown.In order to understand the molecular and functional consequences of the DNMT3AR882H mutation in leukaemogenesis we developed a mouse model (Dnmt3afloxR882H/+), whereby targeting of the Dnmt3a allele does not inactivate the native locus. This allows conditional expression of the mutant protein within haematopoietic stem and progenitor cells that have developed in the presence of normal levels of Dnmt3a. Dnmt3afloxR882H/+ mice were crossed with mice carrying an interferon-inducible Cre recombinase allele (Mx1-Cre). Upon Cre induction with pIpC the endogenous Dnmt3a exon 23 (last exon), was efficiently replaced by the human exon 23 carrying the R882H mutation. This was confirmed at both the DNA and the RNA level. In order to investigate the molecular effects of Dnmt3aR882H, we analysed pre-leukaemic haematopoietic cells (in mice 5-6 weeks post Cre-induction). We observed that Dnmt3aR882H/+ mutant bone marrow (BM) cells had markedly enhanced self-renewal potential as manifested by increased serial replating in cytokine supplemented methylcellulose media in comparison to WT mice. We next examined the relative haemopoietic reconstitution efficiency of Dnmt3aR882H/+ BM cells in a competitive setting. Dnmt3aR882H/+ BM (CD45.2) cells were mixed with WT competitor BM (CD45.1) cells at a ratio of 1:2 and transplanted into lethally irradiated recipient mice (CD45.1/45.2). The analysis of blood chimerism in recipients indicated an increased contribution from Dnmt3aR882H/+ in comparison to WT cells, indicating the enhanced ability of Dnmt3aR882H/+ BM cells to engraft and reconstitute recipient mice. Currently, we are addressing the role of Dnmt3aR882H in the spontaneous leukaemia development alone as well as in rational combinations with other mutations present in human AML.Additionally, to elucidate the molecular mechanism underlying the striking haematopoietic phenotypes ofDnmt3aR882H/+ we have performed analyses of DNA methylation and hydroxymethylation patterns in the haematopoietic progenitor compartment (lineage negative cell population) of Dnmt3aR882H/+ and WT mice, using reduced representation oxidative bisulfite sequencing (RRoxBS) in the pre-leukaemic setting. Moreover, due to cross-talk between Dnmt3a mediated DNA methylation and other chromatin states, we have analysed Dnmt3aR882H-associated changes in histone tail modifications, using ChIP-Seq. As the DNA and histone modifications associated with Dnmt3aR882H are likely to lead to alter global gene expression, we also performed RNAseq analysis in Dnmt3aR882H/+ and WT mice.Currently, we are conducting an integrated analysis, coupling DNA and histone modifications to aberrant gene expression and Dnmt3aR882H-dependent phenotypes. We predict that this work will provide a detailed understanding of the molecular and cellular effects of DNMT3A-R882H and in so doing reveal rational therapeutic approaches for patients with this common and aggressive subtype of AML. DisclosuresNo relevant conflicts of interest to declare.

Immune Reconstitution after Adoptive Infusion of BPX501 Cells (donor T cells transduced with iC9 suicide gene) in Children Given Alpha/Beta T-Cell Depleted HLA-Haploidentical Hematopoietic Stem Cell Transplantation (haplo-HSCT): Preliminary Phenotypic and Functional Results of a Phase I-II Trial

Background: Immune recovery is crucial for patients treated with allogeneic HSCT and in particular of those receiving a T-cell depleted haplo-HSCT. We recently developed a novel method of graft manipulation based on physical elimination of α/β T cells and B-lymphocytes for preventing graft-versus-host disease (GvHD) and EBV-related lymphoproliferative disorders, respectively. Thanks to this approach, we successfully conducted a prospective trial in children with malignant or non-malignant disorders (ClinicalTrial.gov identifier: NCT01810120). Although patients enrolled in this trial had faster immune recovery and lower incidence of infections than those given haplo-HSCT after infusion of positively selected CD34+ cells, reconstitution of adaptive T-cell immunity remains suboptimal. We therefore designed a phase I/II trial aimed at testing the effect on post-transplant immune recovery of adoptive infusion (within 14 + 4 days after transplantation) of BPX-501 cells in children given haplo-HSCT after depletion of α/β T and B cells (ClinicalTrials.gov identifier: NCT02065869).Patients and methods: As of July 25th 2015, 23 children have been infused with BPX-501 cells. The 9 children included in the phase I portion of the study were given 2.5x105, 5x105, and 1x106 BPX-501 cells/kg, respectively, while the 14 included in the phase II received 1x106 BPX-501 cells/kg. This analysis refers to the 16 patients with a minimum follow-up of 90 days; 7 children had acute leukemia and 9 non-malignant disorders. Basic phenotype of circulating lymphocytes was assessed by flow cytometry on fresh heparinized peripheral blood samples at 10, 20, 30, 60, 90, 120 and 150 days post haplo-HSCT, respectively. The following antibodies were used: anti-TCRαβ FITC/anti-TCRγδ PE/anti-CD3 PerCP-Cy™5.5 (WT31, 11F2, SK7), anti-CD4 APC Cy7 (RPA-T4), anti-CD19 BV 510 (SJ25C1), anti-CD3 BV 421 (UCHT1), anti-CD56 PeCy7 (B159), anti-CD16 APC (B73.1), anti-CD8 APC (RPA-T8) from BD Biosciences (San Diego, CA, USA). Antigen-driven activation of peripheral mononuclear cells was evaluated by standard lymphoproliferation assay (LPA) with 3H-thymidine pulsing on day 4 and harvesting 18 hours later. Antigens included PHA or CMV, EBV and AdV whole viral lysate. Results were scored positive with stimulation indexes (SI) >10 for PHA and >3 for viral antigens.Results: None of the patients died from transplant-related complications. Chimerism analysis investigated through short tandem repeats showed that in all but 4 patients, cells were of donor origin before the infusion of BPX-501 cells. In the 4 patients, there was a reversion to complete donor chimerism after infusion of BPX-501 cells. At early time points after haplo-HSCT, gδ T cells predominated over αβ T lymphocytes; subsequently, this latter population became the more largely represented. The number of both CD3+ T lymphocytes and of BPX-501 cells is shown in Panel A of Figure 1, reconstitution of whole T cells in historical children given haplo-HSCT after depletion of α/β T cells is also shown. The number of CD3+ T lymphocytes reached greater than 0.5x109/L 2 months after infusion of BPX-501 cells. Remarkably, while usually immune recovery after transplantation is characterized by prevalence of CD8+ cells, in our patients the physiological predominance of CD4+ lymphocytes was maintained (Panel B of Figure 1. Reconstitution of natural killer cells (NK) is shown in Panel C of Figure 1. As compared to patients receiving CD34+ selected cell haplo-HSCT, children included in this study had a faster reconstitution of mature KIR+/NKG2A- NK cells. Serum levels of IgA and IgM over time are shown in Panel D of Figure 1: there was a recovery of newly synthetized Ig at 3 months. The analysis of the function of T cells showed that the proliferative response to a polyclonal mitogen or to CMV lysate was comparable to that of a healthy control in 50% of patients as early as day + 60 after haplo-HSCT and BPX-501; on day +150, all patients reached a normal SI. Response to both EBV and AdV antigens was slightly delayed, but progressively improved over time (see also Figure 2).Conclusions: Overall, these data indicate that infusion of BPX-501 cells is able to accelerate the recovery of adaptive T-cell immunity since these cells, once infused, expand in vivo and persist over time, potentially contributing to protect patients from infections. [Display omitted] [Display omitted] DisclosuresMoseley:Bellicum Pharmaceuticals: Employment, Equity Ownership.

ASXL1-Mutated Donor HSCs Evolved into FLT3-ITD Positive AML in the Unrelated Donor and FLT3-ITD Negative AML in the Recipient after Transplant

It has been shown that hematopoietic stem cells (HSCs) acquire somatic mutations over time, most commonly involving DNMT3A, ASXL1, and TET2 genes. These mutation-bearing stem cells contribute to normal blood development, harbor a selective growth advantage compared to their non-mutated normal stem/progenitor counterparts, lead to the development of clonal hematopoiesis, and can subsequently gain additional mutations that confer full malignant potential. Here we report an unusual two cases of genetically distinct AML arising from a single pre-leukemic ASXL1 L866X mutant clone.A 28-year-old female was diagnosed with acute B lymphoblastic leukemia by the classical morphology and immunophenotype. Cytogenetic evaluation showed a normal 46, XX, female karyotype. Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) from a matched unrelated donor, a 33-year-old male, was performed. Donor peripheral blood derived HSCs were mobilized and administered to the recipient. On the day +50 after Allo-HSCT, cytogenetics showed a donor derived 46, XY, male karyotype. Consistent with this, chimerism analysis showed 100% donor chimerism . Twenty months after Allo-HSCT, she relapsed with AML with monocytic differentiation. Immunophenotyping revealed the blasts were positive for CD34, CD117, HLA-DR, CD13, CD33, CD64, CD14 and MPO, and were negative for B-cell or T-cell markers. Cytogenetics showed a normal 46, XY, male donor-derived karyotype. Engraftment study continued to show complete donor chimerism in the bone marrow. Molecular study revealed the ASXL1 L866X mutation. Notably, the ASXL1 L866X mutation was not only found in DNA obtained from the recipient at the time of the original diagnosis of donor cell leukemia (AML), but also found in DNA obtained on day +50 after allo-HSCT when the recipient showed full donor engraftment as well as successful complete remission. Moreover, this ASXL1 L866X mutation was also observed in DNA from the healthy donor at the time of donation of peripheral HSCs. In contrast, this ASXL1 L866X mutation was not observed in DNA obtained from recipient specimens at diagnosis or during complete remission of acute B-lymphocytic leukemia before allo-HSCT. Therefore, these data confirmed that this ASXL1 L866X mutation originated from donor HSCs . Except for the ASXL1 L866X mutation, we did not find mutations in CEBPA, DNMT3A, ETV6 (TEL), FLT3-ITD, FLT3-TKD, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, PHF6, PTPN11, RUNX1, TET2, or TP53 genes in the recipient at diagnosis, remission, or relapse of donor cell leukemia.Eighty-four months after donation peripheral HSCs, the previously healthy donor also developed AML. The blasts were positive for CD34, CD117, HLA-DR, CD13, CD33, CD123, CD38, and MPO, and were negative for B- and T-cell markers. Cytogenetic analysis revealed 46, XY, male normal karyotype. The ASXL1 L866X and FLT-3/ITD mutation were identified in leukemia cells, but were not detected in his germline fingernail DNA. After four cycles of induction chemotherapy, he received HLA-identical sibling Allo-HSCT from his brother for salvaged therapy. He acquired complete donor chimerism on day +60 and FLT-3/ITD-positive cells disappeared. Two months later, the leukemia relapsed and the patient died from the disease.The fact that 84 months after donation peripheral HSCs, the healthy donor,carrying the same ASXL1 mutation, was diagnosed AML with additional new gene mutation, FLT-3/ITD, was strongly supported the most recent theory that mutation acquisition occurs in functionally normal HSCs and clonal evolution of pre-leukemic HSCs precedes human AML. Moreover, our findings provide in vivo evidence to support a clonal expansion human model in which ASXL1 is an early initiation mutation while FLT3 is a subsequent driver mutation for abnormal HSCs clonal expansion and malignant transformation. DisclosuresNo relevant conflicts of interest to declare.

Proof of Benefit in Multiple-Cord Blood Transplantation Evidenced By Early Hematopoietic Reconstitution

Introduction:Umbilical cord blood (UCB) serves as a suitable donor source in hematopoietic cell transplantation. However, the initial time to engraftment in UCB transplantation (UCBT) is delayed because of its insufficient graft cell numbers, which often limits its use in UCBT. According to the report by Japanese UCB bank, the majority of UCB units remain unused clinically because of their low graft cell doses. Therefore, we sought to develop a new strategy to improve outcomes of UCBT by using multiple (more than 3) units to augment graft cell doses. We reported at the ASH meeting 2014 that an early hematopoietic recovery in lethally irradiated mice was observed by combining hematopoietic stem/progenitor cells (HSC/HSPCs) derived from multiple allogeneic mouse strains. Herein to establish a clinically relevant procedure, we provide a proof of benefit of this approach using human UCB cells in xenotransplantation models.Methods:We initially subjected a single UCB (HLA-A2 negative) to CliniMACS Prodigy®(Miltenyi Biotec, Bergisch Gladbach, Germany) to acquire mononuclear cells (MNCs). Obtained MNCs were further processed for isolation of either CD34+ cells or CD3+ T cells using microbeads techniques (Figure 1, Unit A). We next combined 3 frozen UCB units (Units B-D, all HLA-A2 positive) into one bag after thawing and then applied them to CliniMACS Prodigy®. Mixed CD34+ cells were purified from obtained MNCs. These test CD34+ cells were transplanted into sublethally-irradiated immunodeficient mice. Mice in the first group received single unit CD34+ cells alone (Unit A, G1 in Figure 2, 3.2 x 104 cells). The additive effect on human cell engraftment was tested in mixed CD34+ cells by transplanting them (Units B-D, G2, 9.6 x 104 cells) together with the single unit CD34+ cells (Unit A, 3.2 x 104 cells). Another cohort of mice (G3) received Unit A CD34+ cells at a dose 4 times more than that in G1 (1.28 x 105 cells). We also tested whether the inclusion of CD3+ T cells (2 x 105 cells) derived from one UCB (Unit A) favored formation of single donor chimerism. Detailed donor cell chimerism was determined in bone marrow over time by flow cytometry analysis, using HLA subtypes as indicators of donor origins.Results:As formerly reported, in a multiple-donor transplantation model using mouse bone marrow cells, combined multiple units of allogeneic HSC/HSPCs were shown to protect lethally-irradiated recipient mice, and to accelerate the recovery of granulocytes, hemoglobin and platelets when infused additionally with a single graft that was composed of whole bone marrow cells. Single donor chimerism was achieved long-term in these mice. Experiments using sorted population of a graft demonstrated that an appropriate dose of T cells should be contained within the primary unit to achieve single donor chimerism through graft versus graft reactions. We then tested whether our strategy was applicable to human UCB cells in xeno-transplantation models.At the first attempt, it was extremely difficult to extract viable CD34+ cells manually from frozen UCB units. To overcome this problem, we employed CliniMACS Prodigy®. Irrespective of HLA type disparities, we eventually succeeded in extracting viable MNCs from multiple UCB units at one time by using CliniMACS Prodigy®. Purified cells were obtained from them using microbeads techniques, with %CD34 and %CD3 of the final test cells being more than 98 % and 96 %, respectively. Up to 9 UCB units were applicable to CliniMACS Prodigy® together at once.As shown in Figure 2, combined multiple UCB units exhibited robust hematopoietic reconstitution. The human cell chimerism in G2 (mixed units) was as high as that observed in G3 (single unit with the same dose). As evidenced by sequential chimerism analysis, inclusion of T cells in one graft achieved single donor chimerism following transient mixed donor chimerism.Conclusions:Our study uncovered that combined multiple UCB units of allogeneic HSC/HSPCs were capable of accelerating hematopoietic reconstitution when manipulated appropriately and added to an otherwise "insufficient dose" setting of transplantation both for mice and human cases. Development of a new strategy of multiple UCB transplantation will open a door for wider application of UCBT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Infusion of Ex Vivo Expanded Allogeneic Cord Blood-Derived Natural Killer Cells in Combination with Autologous Stem Cell Transplantation for Multiple Myeloma: Results of a Phase I Study

Background: Multiple myeloma (MM) is an incurable disease thought to be characterized by immune dysregulation and exhaustion, whereby proliferation of malignant plasma cells is not checked by the native immune system. Long term remissions in some patients after allogeneic stem cell transplant (SCT) suggest a graft versus myeloma effect; however the treatment-related toxicity limits the widespread use of this modality. Allogeneic natural killer (NK) cells are active in various hematologic malignancies and may have a role against MM, without concomitant graft versus host disease (GVHD). Umbilical cord blood is a potential source for allogeneic NK cells and ex vivo expanded umbilical cord blood-derived NK (CB-NK) cells demonstrate activity comparable to that of peripheral blood-derived NK cells. We describe here the results of a phase I, first-in-human study of ex vivo expanded allogeneic CB-NK cells in conjunction with high dose chemotherapy and autologous SCT.Methods: Patients with symptomatic MM who were appropriate candidates for high dose chemotherapy and autologous SCT were eligible. CB units with at least 4/6 match at HLA-A, B and DR were chosen for each patient. When possible, CB units with potential NK alloreactivity were prioritized. On day (-19) CB units were thawed and mononuclear cells (MNCs) were isolated by ficoll density gradient centrifugation. MNCs were cultured in a gas permeable bioreactor with irradiated (100 Gy) K562-based aAPCs expressing membrane bound IL-21 "Clone 9.mbIL21" (2:1 feeder cell:MNC ratio) and IL-2 (100 IU/mL). On day 7, cells were CD3-depleted via immunomagnetic depletion and remaining cells were re-stimulated with aAPC feeder cells and cultured for an additional 7 days. NK cell purity was determined after 14 days of culture. Due to pre-clinical data demonstrating synergy between lenalidomide and NK cells, patients received lenalidomide (10 mg orally daily) from days (-8) to day (-2). Melphalan 200 mg/m2 was given intravenously on day (-7). Freshly expanded CB-NK cells were infused on day (-5). Autologous peripheral blood progenitor cells (PBPC) were infused on day (0).Results: 12 patients have been enrolled thus far with 3 patients each on the following CB-NK cell dose levels: 5 e6 NK cells/kg, 1 e7 NK cells/kg, 5 e7 NK cells/kg and 1 e8 NK cells/kg. 11/12 patients had at least 1 high-risk feature of progressed/relapsed disease (n=7), high-risk cytogenetics (n=2) or International Staging System III disease (n=3). Successful NK expansion to target dose was achieved in all patients with median NK purity (CD56+/CD16+/CD3-) of 98.9% (96.8-99.7). Expanded cells demonstrated cytotoxicity against classic K562 and MM cell line targets. There were no infusional toxicities and no occurrence of GVHD. One patient (1 e8 NK cells/kg) failed to engraft due to a poor PBPC graft quality; this patient was rescued with a back-up autologous PBPC graft. There have been no other significant adverse events and no second primary malignancies. 11/12 patients are evaluable beyond day 100. Best response has been 8/12 nCR or better, 2/12 VGPR and 1/12 PR. 4/12 patients have progressed at a median of 330 days. By DNA microsatellite chimerism analysis, donor CB-NK cells were detected in 2 patients at the 1 e7 NK cells/kg dose and all 3 patients in the 1 e8 NK cells/kg dose, for at least 5 days after infusion. By a more sensitive flow cytometric chimerism assay using HLA class I-specific antibodies for donor or recipient, donor CB-NK cells were detected in 3 evaluable patients at doses of 1 e7 NK cells/kg, 5 e7 NK cells/kg and 1 e8 NK cells/kg for at least 12 days after infusion. Further analysis of these cells indicated persistence of an activated phenotype (NKG2D+/NKp30+) in vivo.Conclusion: CB-NK cells can be activated and expanded to clinical scale. This is the first clinical study of CB-NK cells for MM. When infused in the setting of myeloablative chemotherapy, up to 1 e8 allogeneic CB-NK cells/kg are well tolerated with no infusional toxicities or GVHD. These cells can persist for at least 12 days in vivo and demonstrate an active phenotype. Though clinical data are early, responses are encouraging in this high-risk patient population. Further updated data will be presented at the annual meeting. DisclosuresOff Label Use: Lenalidomide with high dose chemotherapy and autologous stem cell transplantation. Kaur:UT MD Anderson Cancer Center: Employment. Orlowski:Millennium Pharmaceuticals: Consultancy, Research Funding; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Genentech: Consultancy; Onyx Pharmaceuticals: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding; Acetylon: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding; Forma Therapeutics: Consultancy. Cooper:Intrexon: Equity Ownership, Patents & Royalties, Research Funding; ZIOPHARM Oncology: Employment, Equity Ownership, Patents & Royalties, Research Funding. Lee:Cyto-Sen: Equity Ownership; Ziopharm: Equity Ownership; Intrexon: Equity Ownership.

Lineage Specific Chimerism Analysis Reveals That Mixed Donor T-Cell Chimerism Is Common in the Early Post-Transplant Period Following Myeloablative Allotransplants from HLA-Matched but Not HLA-Haploidentical Donors: A Multivariable Analysis of Allografted Patients from a Single Center

Failure to achieve complete donor T-cell chimerism in the early post-transplant period may impair the graft-versus malignancy effect and contribute to a higher risk of disease relapse. Lineage specific chimerism analysis (LSCA) of T-lymphocytes versus myeloid cells is routinely performed following reduced-intensity or non-myeloablative hematopoietic cell allografts. However, many centers do not perform LSCA following myeloablative conditioning. We performed LSCA in all consecutive patients receiving a first T-replete allotransplant using myeloablative conditioning as defined by the CIBMTR (Giralt 2009 BBMT 15;367) between 2/2006 and 2/2015 (n=232; patient characteristics: median age 49 (18-73); Female 50%, Black 19%, Asian 3% white 78%; donor MRD 41%, MUD 39%, Haploidentical 20%; graft PBSC 88%, BM 11.5%, both 0.5%; diagnosis AML 52%, ALL 18%, MDS 11%, CML 11%, NHL 4% MPS1%, CLL1%, HL 1%; DRI low 10%, intermediate 51%, high 29%, very high 10%. Our objectives were to determine the rate of full-donor and mixed T-cell chimerism in the early post-transplant period (d 30 and d 90) , to determine the association of T-cell chimerism with patient, disease and regimen specific factors, and to determine whether early mixed chimerism impacts post-transplant outcomes following myeloablative conditioning. LSCA was performed on peripheral blood using a RoboSep instrument for automated sorting of CD3-positive T-cells and CD33 positive myeloid cells to 96.5-100% and 98.3-100% purity respectively, and short tandem repeat analysis by PCR. Full donor chimerism was defined as > 90% donor derived cells. Probability of achieving full donor T-cell chimerism in evaluable patients on d 30 and d 90 post transplant were 55% and 71%. In contrast the probabilities of achieving full-donor myeloid chimerism were 99.5% and 93.5% respectively. On univariate analysis the following factors were significantly associated with achievement of full-donor T-cell chimerism on d 30: donor type (haploidentical 100%, MRD 39%, MUD 47%, p<0.001), diagnosis (AML 58%, ALL 60%, MDS/MPS/CML 35%, NHL/HL/CLL 90%, p=0.003), conditioning regimen (busulfan based 37%, TBI based 56%, post-transplant Cy 100%, p<0.001), and on d 90: donor type (haploidentical 100%, MRD 56%, MUD 70%, p<0.001), diagnosis (AML 76%, ALL 77%, MDS/MPS/CML 49%%, NHL/HL/CLL 100%, p=0.002), conditioning regimen (busulfan based 56%, TBI based 77%, post-transplant Cy 100%, p<0.001). For multivariate analysis (Table 1) the exact logistic regression method was used to accommodate the nature of the data. The odds ratios (OR) were estimated by exponentiating median unbiased estimates of regression coefficients. Donor type (haploidentical vs other) and diagnosis (MDS/MPS vs AML) were significant factors. Our institutional approach to patients failing to achieve full-donor chimerism by d 90 is to withdraw immnosuppressive therapy in the absence of active GVHD if T-cell chimerism is > 50% or to administer DLI (starting at 1 x 10e6 CD3+ cells/kg) for patients with CD3 chimerism < 50% . Using this approach long term outcomes for patients who failed to achieve full-donor CD3 chimerism by d 30 were not significantly different from those achieving this threshold (2 yr estimated survival -81% vs 67%; disease-free survival 62%% vs 57%; 6 month CI of grade 2-4 acute GVHD 33% vs 41%; 2 yr CI of non-relapse mortality 9% vs 15%, relapse 29% vs 28%; p=NS for all). These data demonstrate that failure to induce early full-donor T-cell chimerism is relatively common following myeloablative allotransplantation using HLA-matched but not haploidentical donors. Although spontaneous improvement can occur, routine LSCA followed by immunologic manipulation of patients with suboptimal chimerism may assist in preventing adverse long term outcomes.Table 1D 30 - OR = odds ratio of achieving full-donor CD3 chimerismFactorEffectOR95% CIP valueDiagnosisALL vs AML0.980.36 - 2.651.000MDS/MPS/CML vs AML0.370.13 - 0.940.035NHL/HD/CLL vs AML5.220.48 - 2700.255DonorTypeMRD vs MUD0.630.29 - 1.350.266Haploidentical vs MUD57.312.0 - ∞<0.001Table 2OR = odds ratio of achieving full-donor CD3 chimerismFactorEffectOR95% CIP valueDiagnosisALL vs AML0.700.20 - 2.490.719MDS/MPS/CML vs AML0.150.04 - 0.48<0.001NHL/HD/CLL vs AML3.870.68 - ∞0.216Donor TypeMRD vs MUD0.350.12 - 0.980.045Haplo vs MUD19.03.64 - ∞<0.001 DisclosuresNo relevant conflicts of interest to declare.

Retrospective Analysis of Minimal Residual Disease-Guided Preemptive Treatment in Patients with AML and MDS - the SAL Study Group Experience

IntroductionDetection of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) in complete hematological remission (CR) offers a potential therapeutic window allowing early interventions in order to prevent overt hematological relapse. Due to the absence of generic markers in AML/MDS, MRD assessment seems to be a complex procedure. As prospective clinical studies are sparse, we initiated a retrospective survey within the SAL (Study Alliance Leukemia) study group and members of the German Cancer Consortium to evaluate the current clinical use of the approach and related clinical outcome of treated patients.PatientsBased on questionnaires we analyzed 84 patients from five clinical centers (pts, m/f=46/38; median age 52 yrs (19-74)) with either AML (n=77) or MDS (n=7) in CR after conventional intensive chemotherapy (CTx; n=23) or allogeneic hematopoietic stem cell transplantation (allo-HSCT; n=61), undergoing preemptive therapy. Mutations, cytogenetic aberrations or donor chimerism analysis for transplanted pts were monitored as MRD markers. Thirty-three pts had a normal and 14 pts - a complex karyotype; 6 pts carried -7 and/or inv(3); other aberrations were detected in 29 pts; in 2 pts no data were available. Among molecular aberrations, NPM1 (n=23) and FLT3-ITD (n=13) were the most frequent, followed by CBFβ/MYH11 (n=8), RUNX1/RUNX1T1 (n=8) and MLL/AF6 (n=3). In 20 pts no mutations were found. For 5 pts data on molecular diagnostics were not available and MRD was assessed by chimerism.ResultsThe median time to MRD positivity after completion of intensive therapy was 12 months (range, 1-97). Subsequent MRD-guided therapy in pts treated only with CTx included hypomethylating agents (HMA, 34%), clofarabine (17%), additional intensive CTx (9%), targeted therapy (gemtuzumab ozogamycin, 9%), low-dose cytarabine (9%), and allo-HSCT (22%). Other treatment strategies included interferon alfa and sorafenib. In transplanted patients the most preferred treatment for the molecular relapse was donor lymphocyte infusion (26 pts, 43%; median number of DLI=3, range, 1-6), alone or in combination (54% with HMA). 32 MRD-based treated pts (43%) did not experience a hematological relapse and were either alive (23 pts, median observation time 953 days, range, 30-3660), or died due to another cause (9 pts) with median survival of 359 days (range, 125-954). In 9 pts without hematological relapse more than one MRD recurrence was observed. In the remaining 57% relapsing patients, median time to hematological relapse was 252 days (range, 24-1161) after MRD positivity. Interestingly, in some pts the hematological relapse was observed in the absence of the known mutation, indicating that the disease probably progressed due to another subclone. Thus, an accurate initial diagnostic is essential, ideally a multiple gene approach and comprehensive follow-up.ConclusionsThe retrospective analysis demonstrates that MRD-guided therapies have already entered routine practice in patients with AML and MDS. The heterogeneous nature of both cancer entities is reflected by the variety of MRD surveillance protocols among clinical centers. Therefore, clinical trials are needed to better define molecular markers and subsets of patients who might benefit from this approach. DisclosuresMayer:Janssen: Research Funding. Thiede:AgenDix GmbH: Equity Ownership. Platzbecker:Novartis: Honoraria; GlaxoSmithKline: Honoraria, Research Funding; Celgene: Honoraria; Amgen, Inc.: Honoraria.