Reduced-intensity Transplantation Research Articles

Haploidentical Natural Killer Cell Therapy as an Adjunct to Stem Cell Transplantation for Treatment of Refractory Acute Myeloid Leukemia.

Refractory acute myeloid leukemia (AML), defined as failure of two cycles of induction therapy at diagnosis or of one cycle at relapse, represents a subgroup with poor outcomes. Haploidentical natural killer cell (NK) therapy is a strategy that is being explored in refractory malignancies. Historically, at our center, patients with refractory AML have been treated with cytoreductive therapy (fludarabine + cytosine + granulocyte colony-stimulating factor ± idarubicin or mitoxantrone + etoposide) followed by 1-week rest and then reduced-intensity transplant with fludarabine + melphalan. We used the same backbone for this trial (CTRI/2019/02/017505) with the addition of CD56-positive cells from a family donor infused 1 day after the completion of chemotherapy. CD56-positive selection was done using a CliniMACS Prodigy system (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by overnight incubation in autologous plasma with 2 micromolar arsenic trioxide and 500 U/mL of interleukin-2. From February 2019, 14 patients with a median age of 29 years (interquartile range [IQR]: 16.5-38.5) were enrolled in this trial. Six were females. Six had primary refractory AML while eight had relapsed refractory AML. The median CD56-cell dose infused was 46.16 × 106/kg (IQR: 25.06-70.36). One patient withdrew consent after NK cell infusion. Of the 13 patients who proceeded to transplant, five died of immediate post-transplant complications while two did not engraft but were in morphologic leukemia-free state (both subsequently died of infective complications after the second transplant). Of the remaining six patients who engrafted and survived beyond 1 month of the transplant, two developed disease relapse and died. The remaining four patients are alive and relapse free at the last follow-up (mean follow-up duration of surviving patients is 24 months). The 2-year estimated overall survival for the cohort was 28.6% ± 12.1% while the treatment-related mortality (TRM) with this approach was 38.5% ± 13.5%. Haploidentical NK cell therapy as an adjunct to transplant is safe and needs further exploration in patients with AML. For refractory AML, post-transplant NK infusion and strategies to reduce TRM while using pre-transplant NK infusion merit exploration.

Intensity of induction regimen and outcomes among adults with Ph+ALL undergoing allogeneic hematopoietic stem cell transplantation

Tyrosine kinase inhibitors (TKIs) are essential for the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and have allowed for effective, low intensity induction regimens including no or minimal chemotherapy. Whether the use of low intensity induction regimens impacts outcomes after allogeneic hematopoietic stem cell transplant (alloHCT) is less understood. We identified consecutive adult patients with Ph+ ALL undergoing alloHCT in first complete remission (CR1) at our center from 2010 to 2021 and examined the impact of pre-transplant induction intensity on outcomes. Among the 87 identified patients, 44 (51%) received low intensity induction and 43 (49%) received induction with high intensity chemotherapy. Patients receiving low intensity induction were older (median age 60 vs. 47 years, p < 0.01). Following induction, measurable residual disease (MRD) negativity by BCR::ABL1 RT-PCR was similar in the low and high intensity induction cohorts (54% and 52% respectively). Receipt of reduced intensity transplant conditioning was not associated with intensity of induction regimen (39% vs. 19% in low vs. high, respectively, p = 0.06). At a median follow-up of 21 months from transplant, there was no difference between low and high intensity induction with respect to 2-year disease-free survival (58% vs. 56%), 2-year overall survival (62% vs. 63%), 2-year cumulative incidence of relapse (9% vs. 17%), and 2-year non-relapse mortality (33% vs. 29%). We also found no difference in outcomes when patients were segmented by both induction and conditioning regimen intensities. Our retrospective analysis suggests that induction intensity does not impact post-transplant outcomes among patients with Ph+ ALL transplanted in CR1.

Late Graft Failure in MDS and Acute Leukemia Patients Receiving Allogenic Hematopoietic Cell Transplantation with Post Transplant Cyclophosphamide As Gvhd Prophylaxis

Introduction: Post-transplant cyclophosphamide (PTCy) is a commonly used prophylactic therapy for graft-vs-host disease (GVHD), particularly in the setting of haploidentical (haplo) transplantation. Despite prior studies showing comparable survival outcomes when compared to HLA matched unrelated donor transplantation (MUD-HCT) using traditional calcineurin inhibitor based GVHD prophylaxis, the rate of graft failure has been reported to be as high as 12-20% in haplo-HCT recipients. In the current study, we report the impact of PTCy on engraftment failure and overall mortality in AML, ALL and MDS patients after haplo vs MUD HCT receiving a uniform PTCy GVHD prophylaxis to determine if excess late graft failure is due to PTCy, donor source or a combination of both factors. Methods: We performed a retrospective cohort analysis using the Center for International Blood and Marrow Transplant Research database among adult patients who underwent first reduced intensity conditioning (RIC) haplo or 8/8 MUD allogeneic HCT between 2011 and 2018 for AML, ALL or MDS with PTCy GVHD prophylaxis previously described by Gooptu et al (Blood 2021). The two comparative cohorts were: 1.) haplo recipients receiving GVHD prophylaxis with PTCy, calcineurin inhibitor/mTOR inhibitor and mycophenolate, and 2.) MUD-HCT recipients receiving GVHD prophylaxis with PTCy, calcineurin inhibitor/mTOR inhibitor and mycophenolate. The population was restricted to patients that initially engrafted after transplant. The primary outcome was incidence of late graft failure, defined as any graft failure following primary neutrophil engraftment. We sought to identify risk factors for graft failure in the two cohorts. Secondary outcomes were overall mortality and its associated risk factors in the haplo vs. MUD-HCT cohorts. Results: A total of 1336 patients met the eligibility criteria (N=60 haplo and N=2 MUD excluded due to a lack of initial engraftment). Of these, 1151 patients received a haplo-HCT and 185 received a MUD-HCT. Patients in the Haplo group were older (61 vs 65), more ethnically diverse (28% vs 5% non-Caucasian), received more bone marrow grafts (45% vs 16%), had older donors (median age 37 vs 28 years old), received predominately TBI/Cy/Flu regimens (86% vs. 32%) and had longer median follow-up (29 vs 25 months) (Table 1). At 2 years, the adjusted probabilities of late graft failure for the haplo-HCT group was 6.5% (95% CI 5.2-8.0) vs 5.9% (95% CI 2.7-10.9) for the MUD (p = 0.785). The 2-year overall survival, however, was significantly lower for the haplo-HCT cohort, 56% (95% CI 53-39) vs 69% (95% CI 61-75) for the MUD-HCT group (p=0.001). Multivariate analysis to factors associated with increased risk of graft failure demonstrated that a diagnosis of MDS, HR 1.98 (95% CI 1.22-3.20, p=0.005), and being transplanted in the earlier time period (2015-2018 vs 2011-2014 HR 0.39, 95% CI 0.24-0.64, p =0.0002). Multivariate analysis to evaluate risk factors associated with overall mortality demonstrated that haplo-HCT, HR 1.46 (95% CI 1.11-1.91, p-value = 0.007), high risk disease, HR 2.31 (95% CI 1.87-2.84, p=<0.0001), HCT comorbidity score of >/= 3, HR 1.34 (95%CI 1.13-1.58, p=0.0007), and a diagnosis of ALL, HR 0.51 (95%CI 0.39-0.67, p=<0.001) were all significantly associated with increased risk. A post-hoc sensitivity analysis to evaluate the effect donor age between donor types on graft failure was performed. Cases and controls were selected where donor age was between 18-49 years old, resulting in a sub-cohort of 1078 patients (898 haplo and 180 MUD). When adjusted for donor age, graft failure did not differ between haplo and MUD HCT HR 1.19(95% CI 0.53 - 2.69, p=0.6731) (Figure 1). Conclusion: In this registry-based analysis of patients undergoing reduced intensity transplant for AML, ALL or MDS using GVHD prophylaxis with PTCy, there was no significant difference in late graft failure rates when utilizing a haplo vs. MUD-HCT with PTCy for GVHD prophylaxis. Overall mortality, however, was significantly higher in the haploidentical donor cohort, as previously reported. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Umbrella Trial in Myeloid Malignancies: The Myelomatch National Clinical Trials Network Precision Medicine Initiative

Introduction: To accelerate myeloid cancer therapeutics, the National Clinical Trials Network (NCTN) is launching the National Cancer Institute (NCI) Myeloid Malignancies Molecular Analysis for Therapy Choice (myeloMATCH) precision medicine clinical trial. Sponsored by NCI and conducted across the entire NCTN system with initial trials to be led by SWOG, Alliance, ECOG-ACRIN, and CCTG, the initiative leverages public-private partnerships with pharmaceutical industry and biotech companies to create an efficient regulatory model incorporating cross-company novel-novel combinations in trials for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). The goal is to create a portfolio of rationally designed treatment substudies onto which patients sequentially enroll over their treatment journey. As patients transition to higher tiers with increasingly lower remaining tumor burden, the focus will be to target residual disease more precisely. Methods: Newly diagnosed patients are enrolled onto the Master Screening and Reassessment Protocol (MSRP) for baseline clinical and laboratory evaluation. Specimens are sent to the Molecular Diagnostics Network (MDNet) with a 72-hour turn-around for patient assignment to an initial treatment substudy via an integrated informatics system developed by the NCI Precision Medicine Analysis and Coordination Center (PMACC). Assignments will be based on algorithms adjusted for prevalence of co-mutations to enhance accrual of rare molecular subsets to specific targeted treatment trials. As shown in figure 1, there are 4 tiers and 5 clinical baskets. Tier-1 is for initial therapy grouped by MDS, younger AML, and older AML. These are typically randomized phase 2 studies testing sensitivity to novel drug combinations with measurable residual disease (MRD) assessment conducted centrally by MDNet. Subsequent therapy occurs in higher tiers. These assignments are made by MDNet/PMACC based on prior treatment substudy outcome. Flow cytometry and duplex sequencing will be employed in Tier-4 clinical trials that will target residual disease. Statistical designs will evaluate the clinical utility of the assays and biomarkers to determine if targeting residual disease confers clinical benefit. Planned activation is quarter 4 of 2022 with the MSPRP, 2-young adult tier-1 studies and 1 tier-2 study. These are testing combinations of azacitidine, venetoclax, CPX351, 7+3 for ELN defined high risk AML, standard risk AML, and in tier-2 the ability to "erase” residual disease after tier-1 treatment. Additional studies in development include agents for mutant TP53, KIT, FLT-3, NPM1, IDH 1/2, higher and lower-risk MDS and a study for reduced intensity transplant and maintenance to include efforts for diverse populations. Launch for these studies is planned for mid 2023. Tier-4 studies to target KIT, IDH, FLT3 and others are in discussion. Discussion: MyeloMATCH is the largest focused investment of infrastructure and researchers ever coordinated by the US Network Groups and NCI that follows patients from diagnosis thorough all treatment in a single disease area. The charge is to rapidly advance therapeutics in myeloid malignancies. MyeloMATCH is designed to efficiently screen and assign patients to precision treatment trials of promising therapeutic combinations. By using early endpoints to identify large activity signals, myeloMATCH will generate data with promising findings for definitive study. The clinical and laboratory data can be interrogated across the initiative to generate hypotheses for additional focused testing. Participants receiving their treatment journey through myeloMATCH will contribute to a unique clinically annotated database with specimens for "omics” serially collected from pre-treatment and through follow up. This will provide a national resource for understanding drug sensitivity and resistance, as well as clonal evolution. In this manner, myeloMATCH aims to reduce the time and investment in failed phase 3 studies, and instead aims to provide high-quality randomized trial data that will enhance selection of phase 3 priorities. We believe this new paradigm for the collaborative conduct of clinical trials may provide meaningful advances for patients with AML and MDS, mentor investigators, and accelerate drug development. Updates and specific treatment-trial designs will be discussed at the meeting. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Reduced intensity hematopoietic stem cell transplantation for accelerated-phase myelofibrosis

Accelerated-phase myelofibrosis, currently defined by circulating blasts 10% to 19%, usually confers very high risk for progression and poor outcome. The outcome of hematopoietic stem cell transplantation for accelerated-phase myelofibrosis has not been evaluated yet. We analyzed the outcome of 349 clinically and genetically annotated patients with primary or secondary myelofibrosis undergoing reduced intensity transplantation, of whom 35 had accelerated-phase myelofibrosis. In comparison with chronic-phase (<10% blasts) myelofibrosis, median leukocyte counts were higher, more patients had constitutional symptoms, and RAS mutations were detected more frequently in the accelerated-phase group. After a median follow-up of 5.9 years, estimated 5-year overall survival was 65% (95% confidence interval [CI], 49% to 81%) vs 64% (95% CI, 59% to 69%) for the chronic-phase group (P = .91), and median overall survival was not reached. In terms of relapse-free survival, estimated 5-year outcome for the accelerated-phase group was 49% (95% CI, 32% to 67%) vs 55% (95% CI, 50% to 61%) for the chronic-phase group (P = .65). Estimated 5-year nonrelapse mortality was 20% (95% CI, 8% to 33%) for the accelerated-phase group vs 30% (95% CI, 24% to 35%; P = .25) for the chronic-phase group. In terms of relapse, 5-year incidence was 30% (95% CI, 14% to 46%) for the accelerated-phase group vs 15% (95% CI, 11% to 19%) for the chronic-phase group (P = .02). Results were confirmed in multivariable analysis and propensity score matching. In conclusion, reduced intensity transplantation showed excellent survival but higher relapse for accelerated-phase myelofibrosis.

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

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

Impact of Peri-Transplant Molecular Mutations on Outcomes of AML Patients Undergoing Fludarabine/Melphalan Conditioned Allogeneic Stem Cell Transplant

Impact of Peri-Transplant Molecular Mutations on Outcomes of AML Patients Undergoing Fludarabine/Melphalan Conditioned Allogeneic Stem Cell Transplant

Immunodynamics of Paroxysmal Nocturnal Hemoglobinuria

In the context of immune-mediated attack in aplastic anemia (AA), PIGA mutations produce a clonally-restricted escape phenotype in an attempt to restore hematopoiesis. Along with PIGA lesions, HLA class I/II mutations may represent an alternative way to render cytotoxic T cell recognition less effective. Microdeletions of 6p21, uniparental disomy [UPD] of 6p and mutations of HLA genes have been previously identified in AA patients. One could stipulate that paroxysmal nocturnal hemoglobinuria (PNH) may be an alternative mode of clonal evolution as opposed to HLA mutation acquisition or instead be a redundant pathway of immune escape co-existing with such lesions. Here, we took advantage of a large cohort (n=92) of patients with hemolytic PNH (granulocytes clone size >20% and LDH>x2.5 ULN) to elucidate the immunodynamics of PIGA/HLA clone(s) (Fig1A).We first used a deep targeted sequencing panel covering HLA classical loci and applied an in-house newly developed pipeline on 62 patients with PNH. Overall, 35% of cases had HLA alterations, which were found at a similar rate in patients studied at onset (33%) and during the course of the disease (32%). These alterations were classified as allelic losses in 15% of cases while majority of mutations were missense (57%) followed by 5‘ or 3‘ UTR (20%), nonsense (15%) and frameshifts (8%) with a median variant allelic frequency (VAF) of 5% (range, 2-95). Of note, the canonical exon 1 HLA B14:02 mutation (c.19C>T, p.R7X) 1,2was found in 2 patients who both had de novo PNH with a granulocyte clone size of 60% at disease onset (Fig1B).To clarify whether the PNH phenotype conveys an absolute resistance from immune attack, one should determine whether in these patients HLA mutations arise indeed within non-PNH cells. Consequently, we also performed HLA sequencing on 9 patients whose samples (n=18) were previously sorted by flow cytometry to enrich for CD59+ and CD59- fractions. HLA mutations were identified in both CD59+ and CD59- fractions at similar rates (44% each) and VAF (5% vs 3%, p=0.3) while no “canonical” exon 1 mutation was found.When looking at associations with disease-specific characteristics, we did not identify any significant differences in terms of burden of HLA events and disease ontogeny (primary vs secondary to AA cases) as well as baseline demographics characteristics (gender, age at presentation), dominant erythrocytes type (II vs III), and thrombotic events. No correlations with specific types of PIGA mutations nor their burden were found in HLA-mutated vs non-mutated groups as also confirmed by the absence of relationship with PNH granulocyte clone size (62% vs 72%, p=0.5). Of note, in HLA mutants the VAFs of HLA lesions were significantly lower than those of PIGA (5% vs 18%, p<0.001) underlying their possible less powerful role in driving immune escape within the context of AA overshooting reactions. To validate these results, we have designed a highly quantitative flow cytometric method for detection of HLA expression (A, B, C for class I; DQ, DR, DP for class II) on CD59+ vs CD59- fractions of CD34+ and CD15+ cells obtained from PNH patients. Preliminary results in 7 patients showed a general downregulation of HLA expression within PNH clones (delta HLA MFI of CD59+ vs CD59- fractions), consistent with our ability to detect HLA mutations also in PNH cells (Fig1C). This preferential down-modulation on PNH cells suggests a possible selection mechanism operating agonistically and in a non-mutually exclusive fashion with the PNH-intrinsic privilege. Moreover, these findings may also represent a byproduct of antigen processing machinery impairment due to proteasomal accumulation of proteins, which would have been GPI-linked and exported to the membrane.In sum, our study indicates that PNH phenotype conveyed by PIGA mutations operates independently from HLA mutations as means of immune escape. In addition, based on these results we stipulate that HLA down-modulation via various mechanisms is operative in the context of the PNH phenotype and the immune privilege of PIGA clones is not absolutely protective. Patients with PNH may have elements of immune suppression. In fact, their responses in the context of reduced-intensity transplant do eliminate hosts PNH clones following transplantation. 3 [Display omitted] DisclosuresPatel: Alexion: Consultancy, Other: educational talks, Speakers Bureau; Apellis: Consultancy, Other: educational talks, Speakers Bureau. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Regeneron: Consultancy.

Endothelial Activation and Stress Index (EASIX) Measured Pre-Transplant Identifies a Subgroup with High Transplant Related Mortality in Patients with Thalassemia Undergoing Stem Cell Transplantation Using Thiotepa-Treosulfan-Fludarabine Conditioning

Allogeneic stem cell transplantation (SCT) is a curative treatment for beta-thalassemia major. The Pesaro risk stratification classifies patients with thalassemia into three groups (class I, II, and III) to predict the transplant-related mortality (TRM) and graft rejection after myeloablative conditioning with busulfan and cyclophosphamide. However, in developing countries, where inadequate chelation therapy prior to transplant is common, most patients would fall under the class III category. There is heterogeneity in the clinical outcomes in this group. Hence, our center introduced a further refinement in this risk stratification, identifying a subset of “class III high risk” as those with age greater than or equal to 7 years and liver size > 5cm (Mathews V et al. BBMT 2007). Subsequently, we showed that the use of a conditioning regimen containing thiotepa, treosulfan, and fludarabine (TTF) along with a peripheral blood stem cell graft led to improved outcomes in this subset with a reduction in early TRM from 46% (with busulfan-cyclophosphamide) to 13% (Mathews V et al. PLoS One 2013). There is still a need to identify predictors of poor clinical outcomes to optimize further the clinical outcomes of these class III high-risk thalassemia patients. Endothelial activation and stress index (EASIX) is a simple biomarker calculated using lactate dehydrogenase, creatinine, and platelet counts. EASIX has been shown to be predictive of overall survival in various settings like GVHD following reduced-intensity transplants for malignancies in adults, veno-occlusive disease, and nonrelapse mortality following SCT (Luft T et al. Lancet Haem 2017; Jiang S et al. Haematologica 2021; Luft T et al. BMT 2020). Here, we evaluated the role of EASIX (measured before conditioning therapy) as a biomarker in predicting early TRM in patients with thalassemia major who have undergone SCT with a uniform conditioning regimen using TTF at our center. During the study period from January 2012 to December 2019, 281 patients with thalassemia major underwent SCT with a uniform TTF protocol at our center. The median age was nine years (range 1 to 25 years). One hundred and nine (38.8%) were females. As per Pesaro classification (with Vellore modification), three (1.1%) were class I, 34 (12.1%) were class II, 134 (47.7%) were class III low risk, and 110 (39.1%) were class III high risk. The stem cell donors were matched sibling (n=218, 77.6%), matched related non-sibling (n=23, 8.2%), or matched unrelated donors (n=40, 14.2%). The stem cell donor was HLA matched in all cases except 21 (7.5%), wherein there was a mismatch at one locus. Five (1.8%) had a bone marrow graft, while others had peripheral blood stem cell grafts. Thirty-eight (13.5%) patients had transplant-related mortality by day 100 (TRM100). The median follow-up of the cohort was 31 months (range 0 to 103 months). EASIX score pre-transplant was available for 184 (65.5%) patients. There was no difference in the rate of TRM100 in patients where EASIX was available compared to those where EASIX was not available (14.7% versus 11.3%, p 0.47). Also, there was no difference in overall mortality rate in patients where EASIX was available compared to those where EASIX was not available (21.2% versus 17.5%, p 0.53). Among patients with TRM100 vis-à-vis those who did not, the median EASIX score was significantly higher (1.09 versus 0.75, p 0.008). We then plotted a receiver operating characteristics (ROC) curve for predicting TRM100 using the EASIX score. The area under the curve was 0.661 (Figure 1a). A cut-off of 0.85 for the EASIX score had a 70.4% sensitivity and 62% specificity for predicting TRM100. The TRM100 for patients with EASIX above 0.85 was significantly higher than those with EASIX less than 0.85 (24.4% versus 7.5%, p 0.003). On multivariable logistic regression analysis, the factors independently predicting TRM100 were pre-transplant EASIX score, pre-transplant ferritin, unrelated donor source, and chimerism at day 60 (Figure 1b). In patients with thalassemia major undergoing SCT using a uniform TTF conditioning, EASIX score measured pre-transplant can identify patients at greater risk for Day100 TRM. [Display omitted] DisclosuresMathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Subclonal Evolution Characterizes MDS Disease Progression Following Allogeneic Stem Cell Transplant

MDS is an oligoclonal disease consisting of a founding clone and one or more subclones derived from the founding clone. Relapsed disease in myeloid malignancies is often characterized by the emergence of a subclone, presumably from selection pressure, following chemotherapy. The contribution of subclone evolution to MDS disease progression following an allogeneic hematopoietic cell transplant (alloHCT) has not been comprehensively studied.We used a combination of exome sequencing, ultra-deep error-corrected targeted sequencing, and copy number analysis to comprehensively describe the clonal dynamics of MDS in response to pretransplant and transplant therapy using 58 serially acquired tumors harvested from 9 patients who all progressed post-alloHCT after receiving either a myeloablative (n=3) or reduced-intensity (n=6) transplant (median time to progression 309 days, range 98-881). We performed enhanced exome sequencing (EES) to define the clonal architecture of 23 tumor samples at the following clinical landmarks (with matched skin as a source of normal DNA): first sampling (usually diagnosis), <2 months pre-alloHCT (where available), and progression post-alloHCT. Somatic mutations were validated in the 23 discovery samples and genotyped in 35 additional samples post-alloHCT, using capture probes targeting all putative single-nucleotide variants (SNV) and short insertions and deletions (indels) identified by enhanced exome sequencing. In addition, ultra-deep, error-corrected sequencing (i.e., barcoded sequencing) was performed on 49 of the 58 tumor samples to provide increased sensitivity to detect low-level mutations. Structural variants (SVs) were identified using exome and FISH data.We found that a subclone expanded or emerged at progression post-alloHCT in all 9 patients. We also observed that mutations (SNVs and/or indels) detected in the founding clone at first sampling always persisted at progression post-alloHCT. In 7 of 9 patients, the expanding/emerging subclone became the most abundant clone at relapse, underscoring the importance of subclonal expansion for driving disease progression. In 6 of these 7 patients (and 7 of 9 overall), the expanding/emerging subclone contained mutations and/or SVs known to be associated with myeloid malignancies. Furthermore, in 4 cases, a rare or minor pretransplant subclone escaped eradication, acquired new mutations or structural variants, and emerged as the most abundant clone at progression. This suggests that minor pre-existing, treatment-resistant subclones can undergo further evolution and drive progression post-alloHCT. In three of these patients, the emergent subclone at progression also gained SVs. In the other patient, the minor subclone, not detected by ultra-deep sequencing at first sampling, emerged during pre-transplant therapy with azacitidine and was characterized by the acquisition of C to G transversions, including coding C to G transversions in the recurrently mutated AML/MDS genes SF3A1 and EPPK1 ; thus the pretransplant therapy shaped the clonal evolution at progression. Overall, SVs were present in the emergent/expanding subclone in 8 of 9 patients. Structural variants were the only new somatic alteration in an expanding/emerging subclone in 5 patients at progression, highlighting the importance of monitoring SVs in post-alloHCT samples, and implicating SVs as late subclonal progression events.We interrogated 35 post-alloHCT specimens to determine if mutations were detectable prior to clinically-defined progression in the 9 patients. In 6 of the 8 patients with available specimens at days 30 and 100 post-alloHCT, mutations were detectable at either or both of these time points. In five instances, standard capture-based sequencing and/or error-corrected sequencing detected low-level mutation burden prior to clinical progression. In all other cases, sequencing results provided independent confirmation of relapse.In conclusion, MDS progression post allo-HCT is characterized by subclonal expansion and mutational evolution, which can be influenced by pretransplant therapy with hypomethylating agents. Although progression is characterized by increasing genetic diversity (including acquisition of SVs) the founding clone mutations are always present at progression, suggesting that tracking founding clone mutations may be useful to detect impending progression/relapse. DisclosuresNo relevant conflicts of interest to declare.

Durable Remission of Refractory and Advanced Stage Mycosis Fungoides/Sezary Syndrome (MF/SS) Utilizing an “Outpatient” Alemtuzumab and Fludarabine-Based Reduced Intensity Allogeneic Peripheral Blood Stem Cell Transplantation (PBSCT)

Background:Advanced MF/SS carries a poor prognosis with most therapies failing to provide a long-term benefit. Allogeneic hematopoietic cell transplantation may result in durable remissions or clinically meaningful downstaging in a subset of patients. However, the risk of transplant related mortality has substantially curbed enthusiasm in the use of conventional allogeneic transplantation for this disease. In this study, we describe a novel allogeneic transplant approach in which a G-CSF mobilized, unmanipulated PBSC allograft from HLA-matched siblings was infused after conditioning with a regimen customized to maximally cytoreduce the disease and to provide the necessary immunosuppression to achieve sustained donor immune engraftment needed for the generation of a graft-vs-tumor effect (GVT). We theorized that this outpatient based reduced intensity transplant regimen would provide the essential tumor-debulking and a strategy to achieve sufficient degrees of donor T-cell chimerism necessary to result in the generation of curative GVT effects in patients with the most advanced forms of MF/SS.Patients and Methods:Between January 2004 and May 2014, 5 subjects (median age of 57, range 27-59) with refractory, advanced stage (III and IV) MF/SS who had failed a median 3 prior treatment regimens (range 2-7) underwent PBSCT following mostly outpatient conditioning with alemtuzumab (total of 193 mg), and fludarabine (total of 125 mg/m2). On day 0, subjects received a G-CSF mobilized unmanipulated PBSC allograft from an HLA identical sibling. CSA alone was used as GVHD prophylaxis. Donor lymphocyte infusions (DLI) were given to all patients to convert T-cell chimerism from mixed to full donor and when necessary to boost a GVT effect.Results:All 5 subjects initially had mixed lympho-hematopoietic chimerism but eventually converted to full donor chimeras in both myeloid and T-cell lineages; full donor T-cell chimerism was achieved at a median 7 months post-transplant (Figure 1). 3/5 subjects never became neutropenic and the 2 subjects who developed neutropenia had rapid neutrophil recovery occurring after 5 and 7 days. No patients developed severe anemia or thrombocytopenia and none required RBC or platelet transfusion support.No patients developed acute GVHD prior to DLI and only one (20%) developed acute GVHD (GI, grade IV) following DLI (the 2nd DLI for this patient). Three subjects (60%) developed limited cGVHD which resolved with short course of steroids.At a median follow-up of 3 years, 4 subjects achieved a complete remission, documented by labs, skin biopsy, and imaging. Remissions were delayed in onset, occurring at median 8 months post-transplant (Figure 1). 3 subjects remain alive in remission 13, 11, and 3 years after transplant. One subject with a heavy smoking history died in remission 3 years post-transplant from a secondary head and neck squamous cell carcinoma, and one subject died 7 months post-transplant from grade IV GI GVHD which followed a 2nd DLI given to treat progressive disease (Table 1).Among those at risk, 3 of 4 subjects (75%) developed CMV reactivation. None of the subjects developed CMV disease or PTLD.Conclusions:Our custom designed conditioning regimen 1) resulted in cytoreductive effects against advanced MF 2) proved to be immunosuppressive enough to allow sustained donor engraftment serving as a platform for subsequent DLIs which lead to curative GVT effects 3) could be performed in the outpatient setting avoiding severe neutropenia and its associated opportunistic infections and the need for platelet and RBC transfusion support. These data establish that long-term cures of even the most advanced refractory MF/SS can be achieved utilizing this alemtuzumab and fludarabine-based reduced intensity allogeneic hematopoietic cell transplant approach. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Double unrelated umbilical cord blood vs HLA-haploidentical bone marrow transplantation: the BMT CTN 1101 trial

AbstractResults of 2 parallel phase 2 trials of transplantation of unrelated umbilical cord blood (UCB) or bone marrow (BM) from HLA-haploidentical relatives provided equipoise for direct comparison of these donor sources. Between June 2012 and June 2018, 368 patients aged 18 to 70 years with chemotherapy-sensitive lymphoma or acute leukemia in remission were randomly assigned to undergo UCB (n = 186) or haploidentical (n = 182) transplant. Reduced-intensity conditioning comprised total-body irradiation with cyclophosphamide and fludarabine for both donor types. Graft-versus-host disease prophylaxis for UCB transplantation was cyclosporine and mycophenolate mofetil (MMF) and for haploidentical transplantation, posttransplant cyclophosphamide, tacrolimus, and MMF. The primary end point was 2-year progression-free survival (PFS). Treatment groups had similar age, sex, self-reported ethnic origin, performance status, disease, and disease status at randomization. Two-year PFS was 35% (95% confidence interval [CI], 28% to 42%) compared with 41% (95% CI, 34% to 48%) after UCB and haploidentical transplants, respectively (P = .41). Prespecified analysis of secondary end points recorded higher 2-year nonrelapse mortality after UCB, 18% (95% CI, 13% to 24%), compared with haploidentical transplantation, 11% (95% CI, 6% to 16%), P = .04. This led to lower 2-year overall survival (OS) after UCB compared with haploidentical transplantation, 46% (95% CI, 38-53) and 57% (95% CI 49% to 64%), respectively (P = .04). The trial did not demonstrate a statistically significant difference in the primary end point, 2-year PFS, between the donor sources. Although both donor sources extend access to reduced-intensity transplantation, analyses of secondary end points, including OS, favor haploidentical BM donors. This trial was registered at www.clinicaltrials.gov as #NCT01597778.

Donor KIR genotype has a major influence on the rate of cytomegalovirus reactivation following T-cell replete stem cell transplantation

AbstractReactivation of cytomegalovirus (CMV) is a common complication following allogeneic stem cell transplantation. Genetic determinants in the host and donor that may influence the rate of reactivation are currently unknown. Viral replication is controlled by T cells and natural killer (NK) cells and these share expression of killer immunoglobulin-like receptors (KIRs). We analyzed whether activatory KIRs carried by the donor influenced the subsequent rate of CMV reactivation in the patient. In transplantations involving siblings where both donor and recipient were CMV seropositive, donors with more than one activating KIR gene were associated with a 65% reduction in CMV reactivation. Multivariate analysis confirmed a significantly reduced risk of CMV reactivation in sibling transplantations where the donor had more than one activating KIR. Reduced-intensity transplantation and graft-versus-host disease grade 2 or higher were associated with an increased risk of CMV reactivation. This observation indicates that activating KIRs play an important role in the cellular control of CMV reactivation.

Conditioning regimen intensity and low-dose azacitidine maintenance after allogeneic hematopoietic cell transplantation for acute myeloid leukemia

Azacitidine (AZA) maintenance following allogeneic hematopoietic cell transplantation (HCT) for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) may reduce relapse risk and improve survival. Given logistic and toxicity-related challenges, identifying subgroups appropriate for this approach is an unmet need. Using data from two centers, we retrospectively compared event-free survival (EFS) and overall survival (OS) of AML and MDS patients who received AZA maintenance (n = 59) with historic controls (n = 90). Controls were selected according to the following criteria: no death, relapse, or Grade III–IV acute GVHD for 100 days after transplant. In multivariable analysis, AZA maintenance yielded significantly improved EFS (p = 0.019) and OS (p = 0.011). Outcomes differed according to regimen intensity. For reduced-intensity transplant, EFS (p = 0.004) and OS (p = 0.004) were significantly improved and equivalent to myeloablative transplant. A significant benefit following myeloablative transplant was not observed. Within the limitation of its retrospective nature, this study suggests that AZA maintenance improves outcomes following reduced-intensity HCT, comparable to myeloablative HCT.

Immune Reconstitution and Infection Patterns after Early Alemtuzumab and Reduced Intensity Transplantation for Nonmalignant Disorders in Pediatric Patients

Hematopoietic stem cell transplantation (HSCT) is a therapeutic option for many nonmalignant disorders (NMD) and is curative or prevents disease progression. Reduced-intensity conditioning (RIC) in HSCT for NMD may reduce regimen-related acute toxicities and late complications. Myeloablation is often replaced by immune suppression in RIC regimens to support donor engraftment. The pace of immune reconstitution after immune suppression by RIC regimens is influenced by agents used, donor source, and graft-versus-host disease prophylaxis/treatment. In a multicenter trial (NCT 00920972) of HSCT for NMD, a RIC regimen consisting of alemtuzumab, fludarabine, and melphalan was substituted for myeloablation. Alemtuzumab was administered early (days -21 to -19) to mitigate major lymphodepletion of the incoming graft and the risk of graft rejection. Immune reconstitution and infectious complications were prospectively monitored for 1-year post-HSCT. Seventy-one patients met inclusion criteria for this report and received marrow or peripheral blood stem cell transplants. Immune reconstitution and infections are reported for related donor (RD) and unrelated donor (URD) transplants at 3 time-points (100days, 6 months, and 1 year post-HSCT). Natural killer cell recovery was rapid, and numbers normalized in both cohorts by day +100. Mean CD3, CD4, and CD8 T-lymphocyte numbers normalized by 6 months after RD HSCT and by 1 year in the URD group. CD4 and CD8 T-lymphocyte counts were significantly higher in patients who received RD HSCT at 6 months and at 1 year, respectively, post-HSCT compared with patients who received URD HSCT. The pace of CD19 B-cell recovery was markedly different between RD and URD cohorts. Mean B-cell numbers were normal by day 100 after RD HSCT but took 1 year post-HSCT to normalize in the URD cohort. Despite these differences in immune reconstitution, the timing and nature of infections did not differ between the groups, presumably because of comparable T-lymphocyte recovery. Immune reconstitution occurred at a faster pace than in prior reports using RIC with T-cell depletion. The incidence of infections was similar for both cohorts and occurred most frequently in the first 100days post-HSCT. Viral and fungal infections occurred at a lower incidence in this cohort, with "early" alemtuzumab compared with regimens administering serotherapy in the peritransplantation period. Patients were susceptible to bacterial infections primarily in the first 100days irrespective of donor source and had no increase in mortality from the same. The overall mortality rate from infections was 1.4% at 1 year. Close monitoring and prophylaxis against bacterial infections in the first 100days post-HSCT is necessary but is followed by robust immune reconstitution, especially in the T-cell compartment.

Introduction and the current status of unrelated peripheral blood stem cells transplantation in Japan

In other countries, unrelated donor peripheral blood stem cell transplantation (PBSCT) is more prevalent than bone marrow transplantation; however, in Japan, it was introduced in 2010 after confirming the safety of donors. PBSCT does not require blood donation, general anesthesia, or frequent bone marrow aspiration of the donor. After PBSCT, numerous hematopoietic cells can prompt blood recovery and engraftment, which has enabled reduced intensity transplantation in elderly patients and patients with concurrent diseases, such as infection. In addition, GVL effect by a large number of donor lymphocytes is expected, however, chronic GVHD is a major concern. When introducing PBSCT in Japan, manuals were drafted considering the short-term safety of donors, and data were collected on the occurrence of long- and short-term adverse events. A randomized trial reported no difference in the survival rate between bone marrow transplantation and PBSCT at 5 years; however, it revealed that QOL was better in the former. PB is a essential transplant source option, and attempts are being made to overcome chronic GVHD. PBSC contains abundant stem cells, progenitor cells, and immunocompetent cells and is indispensable for the development of cell therapy for blood diseases in the future.

Allogeneic Hematopoietic Stem Cell Transplantation for Solid Tumors

Hematopoietic stem-cell transplantation (HSCT) refers to a procedure in which hematopoietic stem cells are infused to restore bone marrow function in cancer patients who receive bone-marrow-toxic doses of cytotoxic drugs, with or without whole-body radiation therapy. Stem cells may be obtained from the transplant recipient (autologous HSCT) or can be harvested from a donor (allogeneic HSCT). Stem cells may be harvested from bone marrow, peripheral blood, or umbilical cord blood shortly after delivery of neonates. The perception of the mechanisms through which malignant cells are eradicated following allogeneic hematopoietic cell transplantation (HCT) has evolved substantially over the past four decades. No longer merely thought of as a means to rescue hematopoietic function following myeloablative conditioning, allogeneic transplantation is now known to be a powerful type of immunotherapy capable of curing patients with other-wise fatal malignant diseases. This conceptual evolution has translated into a diversification of the indications for allotransplants and led to the development of reduced intensity transplant approaches whose beneficial antineoplastic effects occur as a consequence of the transplanted donor immune system. Recently, investigators have begun to test whether non-hematologic malignancies might likewise be susceptible to allogeneic immune attack.

Reduced-Intensity Allogeneic Transplant for Acute Myeloid Leukemia and Myelodysplastic Syndrome Using Combined CD34-Selected Haploidentical Graft and a Single Umbilical Cord Unit Compared with Matched Unrelated Donor Stem Cells in Older Adults

Haplo/cord transplantation combines an umbilical cord blood (UCB) graft with CD34-selected haploidentical cells and results in rapid hematopoietic recovery followed by durable UCB engraftment. We compared outcomes of transplants in older patients with acute myeloid leukemia (AML) or high-risk myelodysplastic syndromes (MDS) who received either HLA-matched unrelated donor (MUD) cells or haplo/cord grafts. Between 2007 and 2013, 109 adults ages 50 and older underwent similar reduced-intensity conditioning with fludarabine and melphalan and antibody-mediated T cell depletion for AML (n = 83) or high-risk MDS (n = 26) followed by either a MUD (n = 68) or haplo/cord (n = 41) graft. Patient characteristics were similar for each graft source except for more minority patients receiving a haplo/cord transplant (P = .01). One half of the AML patients were not in remission. Two-year progression-free survival (PFS), overall survival (OS), and graft-versus-host disease–free relapse-free survival were 38%, 48%, and 32.1% for MUD and 33%, 48%, and 33.8% for haplo/cord transplants (P = .62 for PFS; P = .97 for OS; P = .84), respectively. Acute grades II to IV and chronic graft-versus-host-disease rates did not differ at 19.5% and 4.9% in haplo/cord compared with 25% and 7.4% after MUD (P = .53 and P = .62, respectively). Multivariate analysis confirmed no significant differences in transplant outcomes by donor type. Haplo/cord reduced-intensity transplantation achieves similar outcomes relative to MUD in older AML and MDS patients, making this a promising option for those without matched donors.

Toward Individualizing Conditioning Regimens in Reduced-Intensity Transplantation

The advent of reduced intensity conditioning (RIC) in the 1990's has expanded accessibility to transplant thereby permitting previously ineligible patients to receive such therapy. Consensus guidelines defining dose intensity were subsequently developed (1, 2). Fludarabine combined with intermediate dose busulfan (FB) or intermediate dose melphalan (FM) are the most commonly used RIC regimens. Although these protocols are considered equivalent in terms of myelosuppressive and immunosuppressive properties, there is no prospective comparative data to further guide their use. Therefore, the choice of RIC regimen relies heavily on the preferences and experience of the transplant center.

Therapeutic Cyclosporine Exposure Measured As Area Under the Curve (AUC) Significantly Influences the Outcome after Allogeneic Hematopoetic Stem Cell Transplant (alloHSCT)

Introduction: Prevention of Graft versus host disease (GVHD) forms the mainstay of obstacle in the successful outcome of alloHSCT. Various strategies are employed but combination of calcineurin inhibitors with methotrexate or mycophenolate is the commonest preventative measure used. Cyclosporine is dosed according to trough (C0) levels and the optimum level is defined as between 200 and 300 ng/ml. Daily doses are adjusted to achieve the therapeutic levels. As there can be fluctuations in the daily levels, it was postulated that area under the curve (AUC) extrapolated from daily levels may be more useful to define the optimum cyclosporine exposure for prevention of GVHD. Hence this retrospective analysis was carried out to evaluate if cyclosporine AUC for first 14 days has correlation with the incidence of GVHD.Methods: 549 patients who received allograft and had cyclosporine C0 levels available for first 14 days were included in the analysis. The patient population included 353 males (64.3%) and 196 females (35.7%) with a median age of 44 yr. (range: 16-71). Underlying diagnosis included Ac. Leukaemia (n=319, 58.1%), Chr. Leukaemia (n=42, 7.7%), lymphoma (n=119, 21.7%), myeloma (n=57, 10.4%) or other (n=12, 2.2%).Donor was matched unrelated (n=330, 60.1%), sibling (n=204, 37.2%) or cord (n=15, 2.7%). Stem cell source was PBSC (n=454, 82.7%), BM (n=78, 14.2%), both (n=2, 0.4%) or cord blood (n=15, 2.7%). 415 patients had RIC (75.6%) and 134 had full intensity SCT (24.4%). Alemtuzumab or ATG was used in conditioning in 311 cases (56.6%). The target Cyclosporine AUC with a daily level of 200 was predicted as 2600, 2470 for cyclosporine level of 190 and 2730 for levels of 210.Results: The median AUC for the entire cohort was 3338 (range: 370-6390). Only 56 cases (10.2%) had AUC below 2600. Incidence of AGVHD was significantly higher with use of PBSC (53.7% vs. 21.7%, p<0.001), NHL (66.3% vs. 44.8%, p=0.002) and Alemtuzumab/ATG use (55.9% vs. 40.8%, p<0.001), non-TBI conditioning (57.3% vs. 38.7%, p<0.001) but there was no difference with gender, gender mis-match, intensity of conditioning, CMV match or underlying malignancy. Risk of GVHD was not related to cyclosporine AUC below 2600 (41.2% vs. 50.3%, p=0.2), below 2470 (38.6% vs. 50.3%, p=0.14) or below 2730 (49.4% vs. 49.4%, p=0.99). Similarly incidence of CGVHD was higher with use of PBSC (66.7% vs. 36.6%, p<0.001), Alemtuzumab/ATG (66.2% vs. 53.8%, p=0.003), RIC (64.1% vs. 50.7%, p=0.013) and prior AGVHD (100% vs. 22%, p<0.0001). There was no effect of gender, gender mis-match, underlying malignancy and cyclosporine AUC. AUC did not influence the incidence of AGVHD or CGVHD when the analysis was done separately for full and reduced intensity transplants. The median OS from HSCT was 4.9 yr. (0.01-23.8 yr.). Even though incidence of AGVHD or CGVHD was not influenced by cyclosporine AUC, OS was significantly lower for AUC below 2470 (2.2 vs. 4.9 yr., p=0.0002), below 2600 (2.5 vs. 5.0 yr., p=0.0001) and below 2730 (2.1 vs. 4.6 yr., p<0.0001). By multi-variate analysis, Alemtuzumab/ATG (RR: 1.55, 95% CI: 1.04-2.3, p=0.031) and AUC below 2600 (RR: 1.9, 95% CI: 1.3-2.8, p<0.001) were independently associated with inferior OS. Patients with both the factors had lowest OS (none: 7.5 yr.; one: 5yr., both: 1.1 yr.; p<0.0001).Conclusion: This retrospective analysis had high proportion of cases receiving Alemtuzumab/ATG based GVHD prophylaxis and shows that even though there is no effect of cyclosporine AUC on the incidence of either AGVHD or CGVHD, patients receiving Alemtuzumab/ATG with sub-therapeutic cyclosporine AUC have poor OS. This raises the possibility that in this group of patients it is imperative to have target cyclosporine levels in first 14 days. The duration of target cyclosporine levels before tapering the doses needs to be established to improve OS without increasing the toxicity. DisclosuresSomervaille:Imago Biosciences: Consultancy; Novartis: Consultancy, Honoraria. Bloor:Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria; GSK: Consultancy, Speakers Bureau.