PBSC Grafts Research Articles

Peripheral Blood Grafts Vs Bone Marrow Grafts in Young Acute Myeloid Leukemia Patients with HLA-Matched Sibling Donors Undergoing Myeloablative Conditioning

In allogeneic hematopoietic cell transplantation (allo-HSCT), graft source has been changed from bone marrow grafts (Bone marrow stem cell, BMSC) to peripheral blood grafts (Peripheral blood stem cell, PBSC) mobilized by granulocyte colony-stimulating factor. PBSC transplantation is known to have numerous advantages over BMSC, including faster engraftment, improved survival outcomes, and for the donor, the elimination of invasive marrow harvesting procedure under general anesthesia. However, previous studies regarding graft source included various hematologic malignancies and lacked consistency in conditioning intensity and disease status at allo-HSCT. Still, there are limited data on the comparison of graft sources specifically in patients with acute myeloid leukemia (AML) undergoing myeloablative conditioning. In this background, we conducted an analysis to investigate the impact of graft source on transplant outcomes in AML patients who underwent HLA A, B, C, DR-matched sibling donor (MSD) HSCT with myeloablative conditioning. This study was a retrospective, single-center study for patients with AML who received first allo-HSCT from 2002 to 2022. We included the patients aged < 60 at the time of allo-HSCT, in a state of complete remission (CR) or CR with incomplete hematologic recovery, from a MSD, and underwent myeloablative conditioning. We classified the patients into three groups: the BMSC graft group (BMSC group), the PBSC graft group without antithymocyte globulin (ATG) (PBSC/ATG- group), and the PBSC graft group with ATG (PBSC/ATG+ group). Patients who received both PBSC and BMSC grafts were excluded from the analysis. We examined the differences in overall survival (OS), relapse-free survival (RFS) among these three groups. A total of 480 patients were included in this analysis, with 234 patients in the BMSC group, 66 patients in the PBSC/ATG+ group, and 180 patients in the PBSC/ATG- group. BMSC transplantation was mostly performed before 2010, whereas PBSC transplantation started in 2007, and allo-HSCT with ATG administration were conducted after 2014. In the PBSC/ATG+ group, the median ATG dose was 2.5 mg/kg (Interquartile range, IQR: 1.6-2.5). The BMSC group tended to be younger at the time of HSCT (median 38 years [31-46]) compared to the other groups (median age of PBSC/ATG+ group: 44 years [37-51], PBSC/ATG- group: 46 years [39-51], p<0.01). Of total patients in the three groups, there was only 1 case of neutrophil engraftment failure (in the PBSC/ATG- group) and 5 cases of platelet engraftment failure (3 in the BMSC group and the other 2 in the PBSC/ATG- group). BMSC patients demonstrated superior OS compared to PBSC patients, with the most pronounced difference was observed over PBSC/ATG- group (Figure A). Similar trends were presented in relapse-free survival (RFS) as well. After adjusting for other covariates through multivariate Cox modeling, the difference caused by the graft source remained significant, particularly in terms of RFS (Figure B). The hazard ratios for RFS of PBSC comparing to BMSC were as follows; PBSC/ATG+: 1.64 [1.04-2.58], PBSC/ATG-: 1.95 [1.45-2.63]. In conclusion, Despite the clear differences in allo-HSCT era between groups, even BMSC patients underwent allo-HSCT at an earlier time, patients receiving BMSC grafts showed superior survival compared to those receiving PBSC grafts in young AML patients undergoing MSD myeloablative allo-HSCT. Parallel evaluation of the transplant outcomes between BMSC and PBSC would be crucial for assessing the advantages and disadvantages of PBSC and BMSC grafts in this setting.

Single Center Retrospective Study of Midostaurin Plus Intensive Chemotherapy for Older Patients with Newly Diagnosed FLT-3 Mutated Acute Myelogenous Leukemia

Background: Mutations in FLT3 (FLT3m), either internal tandem duplication (ITD) or tyrosine kinase domain (TKD) are found in approximately 25% of AML patients. The current standard of care for eligible patients is induction chemotherapy with 7 + 3 regimen with the addition of multikinase inhibitor midostaurin, based on CALGB 10603 which enrolled patients up to 59 years of age (Stone et al; NEJM 2017). Quizartinib recently demonstrated improved overall survival (OS) compared to placebo when combined with standard 7+3 induction in patients up to 75 years of age, although the benefit in patients 60 and above was less clear (Erba et al; Lancet Oncol 2023). We sought to review the clinical outcomes among patients 60 years of age or greater at our institution for who received midostaurin with 7+3 for newly diagnosed (ND) FLT3m AML. Methods: We retrospectively reviewed 20 patients, 60 years of age or older who received midostaurin in combination with 7+3 (7+3+Mido) for the treatment of ND-FLT3m AML. Response criteria of CR (complete remission) and CR without hematological recovery (CRi) were defined per standard International Working Group (IWG) AML response criteria. The primary endpoint was defined as composite CR rate (cCR: defined as patients achieving CR +CRi). Minimal residual disease (MRD) assessment was done on post-induction bone marrow aspirate using multiparametric flow cytometric assessment. The secondary endpoints were OS, leukemia-free survival (LFS) among patients with CR/Cri and proportion of patients who underwent subsequent allogeneic hematopoietic cell transplant (alloHCT). Results: Patients' demographic, cytogenetic and molecular features are summarized in Table 1, briefly, the median age was 64 (range 61-75), males (n=12, 60%), de novo AML (n=19, 95%), FLT3 ITD (n=13, 65%), FLT3 TKD (n=7, 35%). The most common consolidation regimen was high dose cytarabine (HiDAC) in combination with midostaurin (n=5, 25%). Grade 3 or higher hematologic toxicity was uniformly seen and the most common grade 3 or higher non-hematologic toxicities were neutropenic fever (19/20; 95%), cardiac disorders (3/20; 15%), and diarrhea (3/20; 15%). Dose reductions/interruption of midostaurin was required in 35% patients (n=7) and one patient died due to induction related mortality. 7+3+Mido yielded an overall response rate of 90% (18/20), with cCR noted in 75% (15/20) and MLFS seen in 15% (3/20) patients. MRD negative remission was documented in 40% (6/15) patients treated (table 2). The median follow up for the entire cohort was 9.3 months and overall survival at 6 months was 70% (14/20). The majority of patients, 70% (14/20) subsequently underwent alloHCT from matched or haploidentical donor. Reduced intensity conditioning (n=12/14; 86%) and GVHD prophylaxis with tacrolimus/ sirolimus prophylaxis (n=8; 57%) was commonly used in patients undergoing alloHCT. Six patients did not proceed to alloHCT due to death (n=4, one each due to sepsis, disease progression, infection, or intracranial bleed), comorbid conditions (n=1) or short follow up. The median OS for non HCT group was 2.3 months (range: 0.6-6.5). Detailed HCT outcomes data was available subset of patients at last data cut off (9/14; 65%). The Median age of patients undergoing HCT was 65 years (range: 62-76). The median KPS and HCT-CI for these patients was 90% (range :80-90%) and 1.5(range: 0-5) respectively. The majority of patients underwent alloHCT in CR-1(90%), all using PBSC graft from donor. The median duration of follow-up from transplant to last contact or death in the nine evaluable patients was 12.1 months. Median OS and leukemia free survival was not reached and 1-year GRFS was 50% (range:14-79%). Five patients (36%) patients received post-transplant FLT3 maintenance with the most common agents being midostaurin (n=2) and gilteritinib (n=2). Conclusion: The addition of midostaurin to intensive 7+3 chemotherapy induction for ND FLT3m AML is feasible and well tolerated in older AML patients. The combination results in high remission rates and the majority of patients are able to proceed to alloHCT.

Combined Cytokine Blocking Therapy (CCBT) Using Basiliximab and Infliximab for Treatment of Steroid-Refractory Graft Versus Host Disease (SR-GvHD)

Background Despite improved advances in graft-vs-host disease (GvHD) prophylaxis regimens and post-allogeneic hematopoietic cell transplant (HCT) supportive care, clinically significant acute GvHD still develops in approximately 30-50% of HCT recipients (Lazaryan A et al. BMT 2016) and often results in increased transplanted-related morbidity and mortality and deterioration of quality of life (Choi J et al. Blood 2012). Standard first-line treatment is high-dose glucocorticoids have been limited including recently approved JAK2 inhibitor (ruxolitinib). The utilization of combined cytokine blockade therapy (CCBT) with the monoclonal antibodies infliximab (a TNF-α inhibitor, INF) and basiliximab (an IL-2 receptor blocker, BAS), has had limited discussion in the literature despite common utilization. The primary objective of this retrospective study was to determine overall response rate (ORR) at days 7, 14 and 28 for CCBT. Secondary outcomes included non-relapse mortality (NRM), and overall survival (OS). Methods 60 patients who met the inclusion criteria were analyzed. Patients included were ≥18 years old and had undergone HCT using any donor or graft source with any conditioning regimen between 2010-2021 at City of Hope Medical Center in Duarte, CA. Patients had steroid refractory aGvHD, according to Mount Sinai Consortium guidelines (Harris AC et al. BMT 2016) and received at least one dose of BAS and at least one dose of INF. Patients with >1 allogeneic transplant, chronic GVHD including overlap syndrome, relapse of primary disease, graft loss, and glucocorticoid treatment for indications other than GVHD were excluded. Treatment BAS was administered intravenously as 20mg doses given first as loading doses on days 1 and 4. Subsequent doses were given weekly starting 7 days after the final loading dose (day 4). INF was administered as 10 mg/kg doses infused weekly. Tacrolimus, sirolimus and cyclosporine dosing and monitoring were performed in accordance with institutional policies. All patients had clinical presentation of SR-GVHD defined as ≥1 of the following: GVHD increasing in stage in any organ or developing in a new organ after 3 days of ≥2 mg/kg methylprednisolone (MSPE) or equivalent, GVHD that has not improved in stage in ≥1 organ after 7 days of ≥2 mg/kg MSPE or equivalent, development of GVHD in a new organ after ≥1 mg/kg MSPE or equivalent for skin GVHD or patients who progress during tapering before a 50% decrease in glucocorticoids is achieved. Results The median age was 53 years (range, 5-78) at time of HCT. Of 60 patients, 65% were males and with 18.3% of male patients receiving grafts from female donor. HCT-CI at the time of HCT was 3 or more in 47% of the patients. Conditioning for those patients was ablative in 43.3% of patients with 76.7% of patients receiving PBSC grafts from mostly MSD/MUD in 75% of patients. Most patients had grade 3 or 4 overall acute GVHD (grade 3: n=35, 58.3%, grade 4: n=20, 33.3%). Median time to start of BAS after start of steroid was 7 days while time to start of INF after steroid was 11 days. Ruxolitinib was initiated in 33.3% (20/60) of patients prior to CCBT. ORR for CCBT therapy at day 7, 14, and 28 were 28.3% (17/60; CR 5.0%), 38.3% (23/60; CR 11.7%), and 38.3% (23/60; CR 23.3%), respectively. Patients who received ruxolitinib prior to CCBT had lower ORR (25%) compared to those who did not (47.5%). 37 patients died-20 of aGVHD, 4 of infection, 1 of sudden cardiac death and 1 of leukemia relapse. OS was 41.7% at 6 months and 36.5% at 12 months (Figure 1A). Cumulative incidence of NRM was 51.7% at 6 months and 55% at 12 months (Figure 1B). Cumulative incidence of any cGvHD was 33.6% at 12 months while it was 30% for extensive cGvHD at the same 12-month interval. In patients with and without ruxolitinib initiated prior to CCBT, OS at 12 months was 30% vs 40%, respectively (Figure 2A) while NRM at 12 months was 65% and 50%, respectively (Figure 2B). Conclusion CCBT has potential efficacy in steroid refractory aGVHD. The observed ORR is reflective of both advanced and highly refractory aGVHD with one-third of patients progressing on ruxolitinib. Given the favorable observed ORR in this difficult to treat population, CCBT is a potentially promising approach and suitable alternative in the second line or as salvage therapy in patients failing Ruxolitinib.

Incidence, Risk Factors, Characteristics, and Outcome of Chronic Graft Versus Host Disease in Children Undergoing Haploidentical Peripheral Blood Stem Cell Transplant With Post-transplant Cyclophosphamide.

Chronic graft versus host disease (cGVHD) is a major cause of morbidity postallogeneic peripheral blood stem cell transplant (PBSCT). There is paucity of literature describing incidence, risk factors, characteristics, and outcome of cGVHD in children undergoing haploidentical PBSCT with post-transplant cyclophosphamide (PTCy). Here, we describe our experience from our center regarding the same. All children who underwent haploidentical PBSCT with PTCy between January 2016 and December 2021 at our center and survived beyond day+100 post-transplant were included in this retrospective study. Conditioning regimens used were: Thiotepa-Fludarabine-Cyclophosphamide with 2Gy single fraction total body irradiation, Thiotepa-Busulfan-Fludarabine, Fludarabine-total body irradiation and Fludarabine-Melphalan. Peripheral blood was used as stem cell source in all patients. GVHD prophylaxis was PTCy 50mg/kg on day +3 and +4, Mycophenolate mofetil and Calcineurin inhibitors. Clinical and laboratory data was electronically retrieved and analyzed based on National Institute of Health Consensus Criteria-2014 at regular intervals. Impact of various patient, donor, and transplant-related factors on development of cGVHD were analyzed. Incidence of relapse, event free survival (EFS) and overall survival (OS) were calculated and compared between cGVHD and no cGVHD groups. Patients with rejection were excluded from risk factor analysis for cGVHD but were considered for survival analysis. Fifty-one children included in this study. Median age of transplant of our cohort was 7.5 years with male:female=1.6:1. Eight patients had rejection with autologous recovery. History of acute GVHD (aGVHD) was present in 15/51 (Grade III to IV in 7/51). cGVHD developed in 19/51 patients (mild-9/51, moderate-6/51, and severe-4/51). Skin was the most common organ involved (100%) followed by gastrointestinal tract (47.4%), liver (36.8%), eyes (21%), lungs (21%), mouth (15.7%), and joints (5.2%). Advanced donor age (>30y) and previous aGVHD were found to be significantly associated with increased risk of developing cGVHD. At last follow-up, complete response and partial response of cGVHD was seen in 6/19 and 4/19 patients, respectively. Overall mortality was 15/51 (cause of mortality was relapse of cancer 8/15, cGVHD-3/15, other 4/15). EFS and OS of full cohort was 55% and 70.6%, respectively. Compared with patients without cGVHD, patients with cGVHD demonstrated a lower relapse (18.2% vs. 40%, P =0.2333), higher EFS (68.4% vs. 53.1%, P =0.283), and higher OS (73.7% vs. 68.8%, P =0.708). Incidence of cGVHD was high in children undergoing haploidentical PBSCT with PTCy. Other than PBSC graft source; donor age and previous aGVHD were the risks factors for development of cGVHD. Patients with cGVHD had lower incidence of relapse translating into better survival but this difference was not statistically significant.

Cryopreservation Changes the Immune Effector Cell Composition of Peripheral Blood Stem Cell Grafts: An Analysis from the DKMS and NMDP Graft Composition Study

The impact of the cellular composition of PBSC grafts on the immune reconstitution as well as graft-versus-host and graft-versus-leukemia effects after allogeneic hematopoietic cell transplantation (alloHCT) is only poorly understood. DKMS and NMDP joined forces to comprehensively characterize the cellular composition of 2,000 PBSC products using a 34-color flow cytometry panel to define immune effector cell subsets of interest and hematopoietic stem cells. In parallel, we have created a biobank of unstimulated donor PBMC and PBSC graft samples for future analyses. As part of this larger effort, we investigated the effect of cryopreservation on the cellular composition of PBSC products. Cryopreservation of PBSC grafts was used frequently during the COVID19-pandemic and facilitated more flexible timelines for transplantation. Still, one third of the US patients and 15% of the German patients continue to receive cryopreserved PBSC grafts. Ongoing studies are investigating the impact of cryopreservation on immune-effector cell function and the outcome of hematopoietic stem cell transplantation. Several observational studies have reported delayed neutrophil and platelet engraftment with cryopreserved products. More recently, less chronic GVHD was reported for patients who had received cryopreserved PBSC products. Cryopreservation affects the cellular composition and hence has the potential to change immune effector cell functions of the graft, but available data on the alterations of cell population frequencies are scarce. We analyzed the cellular composition of 20 fresh grafts and compared them to their frozen and thawed counterparts. Cryopreservation of product samples followed routine clinical procedures using a 10% DMSO containing cryo-medium and a controlled rate freezing. After thawing at 37°C, cells were washed, stained with a 34-parameter phenotyping panel, fixed and analyzed immediately on a full spectrum flow cytometer. Following spectral unmixing and manual spillover correction, proportions of immune cells were assessed using manual gating. First, we investigated the relative frequencies of 10 non-overlapping individual cell populations relative to viable CD45+ cells, including several lymphocyte populations, monocytes, granulocytes, hematopoietic stem cells (HSC) and dendritic cells. Granulocytes showed the largest decrease after cryopreservation and thawing (18.2 % vs 3.5 %; p<0.001). The loss of granulocytes results in an increase of relative frequencies of many other immune cell populations including monocytes (35.4 % vs 44.7 %; p=0.018), B cells (5.8 % vs 9.6 %; p=0.006), and HSC (1.0 % vs 1.5 %; p<0.001). We therefore also calculated changes relative to the viable non-granulocyte cells. By doing so, a significant relative increase of cells was observed for HSC (1.2 % vs 1.6 %; p<0.001), whereas monocyte and B cell frequencies changed only marginally. This suggests that HSC are more resilient to freezing than other cell types. When comparing the proportions of 40 other immune cell (sub-) populations relative to their parent population in the gating hierarchy, we found a profound and significant decrease of the relative frequencies for CD16+ myeloid dendritic cells (mDCs, 68 % vs 30 % of all mDCs; p<0.001) and granulocytic myeloid derived suppressor cells (gMDSCs, 2 % vs 1 % of all MDSCs; p<0.001). In an independent set of analyses performed at Roswell Park Comprehensive Cancer Center, Buffalo, comparable results were obtained with a significant increase of HSC, a significant decrease of CD16+ mDCs, and a trend of a decrease of gMDSCs. As CD16+ mDCs may have a role in the development of chronic GVHD after alloHCT, this decrease may be meaningful. In summary, we found that cryopreservation of PBSC allografts resulted in a decreased relative frequency of granulocytes, and CD16+ mDCs, as well as an increased relative frequency of HSCs. The results provide some insight into the impact of cryopreservation on PBSC graft composition and warrant further efforts to explore the potential mechanisms for the differential outcomes observed using fresh vs cryopreserved PBSCs.

Impact of Allograft T-Cell (CD3) Dose on Outcomes When CD34 Cell Dose Is Capped: Analysis of 811 Consecutive Allogeneic PBSC Transplants from a Single Center

G-CSF mobilized PBSC allografts contain higher T-cell doses than bone marrow grafts and have been associated with a significantly higher risk of chronic GVHD. Higher CD34+ cell content in PBSC grafts has also been shown to predict higher rates of chronic GVHD and associated mortality 1,2 To mitigate this risk our center adopted a policy of capping CD34+ cell dose at 5 x 10e6/kg recipient weight for unmanipulated PBSC grafts. For this study, we assessed the effect of T-cell (CD3) dose administered under this capped CD34 strategy, on GVHD and other transplant outcomes. Consecutive first PBSC allografts for hematologic malignancy performed using the CD34 cap strategy between 2010 and 2022 (n=811) were included in this analysis. GVHD prophylaxis was tacrolimus/methotrexate for MRD and MUD and post-transplant cyclophosphamide based for Haplo. Patients receiving anti-T cell serotherapy or ex-vivo T-depletion were excluded. GVHD was uniformly assessed and graded by a dedicated practitioner for all cases. Patient characteristics were: median age 55 (18-80); 57% male; race -white 74%, black 21%, Asian 5%; diagnosis AML 38%, ALL 18%, MDS 18%, NHL 10%; donor type haplo 42%, MRD 32%, MUD 26%, %; high/v. high 25%, low/intermediate 73%; HCT-CI (0-2) 43%, (>3)57%, regimen - myeloablative 48%, RIC 31%, NMA 21%). Median follow-up for survivors was 64 (16-151) months. Median CD34 and CD3 doses infused were 5.0 x 10e6/kg (min 1.59) and 17.3 x 10e7/kg (range 1.2-83.3) respectively. Median CD3 dose infused were higher with female donors (20.5 vs 15.5 x 10e7/kg, p<0.001) and older donors (18.6 vs 15.9 x 10e7 for donors aged >38 vs <38, p<0.001). The effect of Infused CD3 dose was assessed in terciles (ranges 1.2-13.5, 13.6-22, 22.1-83.3 x 10e7/kg). Median peripheral blood donor CD3 chimerism was 100% for all CD3 dose terciles at d 30, through 180 post-transplant. On univariate analysis - estimated 3-year survival, DFS, NRM, relapse and acute GVHD incidence were not significantly different between the low, middle and high CD3 terciles. In contrast, the cumulative incidence (CI) of moderate-to-severe chronic GVHD at 3 years was significantly higher in the highest vs lowest tercile (36.8% vs 28.6%, p=0.042) (Fig 1), while severe chronic GVHD (14.5% vs 13%) was not significantly different. On stratified Cox multivariable analysis comparing the highest to the lowest CD3 dose terciles - survival, DFS, NRM, relapse and acute GVHD were not significantly different. Moderate-to-severe chronic GVHD was significantly higher in the highest versus lowest tercile (HR 1.53, p=0.005). In order to address specific effects of CD3 dose for particular donor groups, we analyzed incidence of chronic GVHD by CD3 dose terciles separately for the three donor types (MRD n=258, MUD n=210 and Haplo n=343). Whereas the CI of moderate-to-severe chronic GVHD was not significantly different for lowest versus highest CD3 tercile in MRD and Haplo transplant recipients, it was significantly higher for MUD recipients (50% vs 26%, p=0.008, GRFS 29% vs 9%, p=0.005). On multivariable analysis, risk of moderate-to-severe chronic GVHD was significantly higher for the highest versus lowest CD3 dose terciles (HR 2.62, p=0.002) and for the middle vs lowest terciles (HR1.77, p=0.043) in MUD patients (Fig 1). In contrast, CD3 dose had no significant impact on chronic GVHD for MRD and Haplo recipients. This large retrospective analysis of PBSC allografts using a CD34 cap (<5 x 10e6/kg) shows that this approach produced infused CD3 doses ranging between 1.2 and 83.3 x 10e6 /kg. The median and upper limit of infused CD3 dose was lower than previously reported by studies with no CD34 cap 2,3. CD3 dose had a significant impact on the rate of moderate-to-severe chronic GVHD in MUD recipients. A prospective comparison of capped versus uncapped CD34 dosing may help determine the value of this strategy in mitigating GVHD risk of PBSC grafts. These data suggest the need for more aggressive GVHD prophylaxis in patients infused with higher CD3 doses in MUD patients. 1. Mohty M, Bilger K, Jourdan E, et al. Leukemia. 2003;17(5):869-875. 2. Zaucha JM, Gooley T, Bensinger WI, et al. Blood. 2001;98(12):3221-3227. 3. Saad A, Lamb L, Wang T, et al. Biol Blood Marrow Transplant. 2019;25(9):1875-1883.

Busulfan, fludarabine, and melphalan are effective conditioning for pediatric and young adult patients with myeloid malignancies underdoing matched sibling or alternative donor transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) remains a curative option for patients with high-risk myeloid malignancies. We present our 10-year experience (October 2012 to October 2021) of consecutive allo-HCT in patients with myeloid malignancies treated on the pediatric HCT service and conditioned with myeloablative targeted dose-busulfan (BU), fludarabine (FLU), and melphalan (MEL). Twenty-three children, adolescents, and young adult patients (CAYA) (median age 15.4years) with acute myeloid leukemia (AML, n = 17), myelodysplastic syndrome (MDS, n = 4), or chronic myeloid leukemia (CML, n = 2) underwent allo-HCT post-BU-FLU-MEL. Four patients had treatment-related AML/MDS. Donor/stem cell source was matched sibling donor (MSD) PBSC (n = 7), matched unrelated donor (MUD) PBSC (n = 2), umbilical cord blood (UCB) (n = 3), or haploidentical-BMT (n = 11). Risk stratification was low (n = 2), intermediate (n = 15), high (n = 3), and very high risk (n = 1). The two patients with CML had failed tyrosine kinase inhibitor therapies. With a median follow-up of 41.6months, the relapse rate is only 4.5% with an overall survival (OS) 100%, progression-free survival (PFS) 95.5%, and graft-versus-host-free-relapse-free survival (GRFS) 67.8%. The donor source and the acute graft-versus-host disease (GvHD) prophylaxis regimen significantly impacted grade II-IV aGvHD 66.7% versus 19.2% (p = .039) and chronic graft-versus-host-disease (cGvHD) 66.7% versus 0% (p = .002) in the patients receiving MSD or MUD PBSC compared to haplo-BMT, respectively, resulting in improved GRFS in haplo-BMT, 83.3% compared to 40% matched donor peripheral blood stem cell transplant (PBSCT) (p = .025). Our results demonstrate that BU-FLU-MEL is efficacious conditioning for disease control in young patients with myeloid malignancies undergoing MSD or alternative donor allo-HCT, but in the setting of PBSC grafts with cyclosporine A-methotrexate (CSA-MTX) GvHD prophylaxis, it results in an unacceptably high incidence of GvHD.

HLA Evolutionary Divergence (HED) Informs the Effect of HLA-B-Loci Mismatch on Relapse and Disease Free Survival after Haploidentical Hematopoietic Cell Transplantation (Haplo) with Post-Transplant Cyclophosphamide (PTCY)

The Success of allogeneic Hematopoietic cell transplantation (HCT) is dependent on a potent graft versus tumor (GVT) benefit. GVT relies on tumor-associated antigens TAAs that are presented to donor T cells via human leukocyte antigens HLAs. The high diversity of HLAs allows for presentation of immense repertoire of peptides including TAAs. HLAs are polymorphic and each allele presents a different repertoire of peptides referred to as the immunopeptidome. The HLA evolutionary divergence (HED) between alleles of a single individual can dictate the ability to present TAAs. Patients with higher HED can present a more diverse immunopeptidome, including TAAs and potentially have a benefit of higher GVT after allogeneic HCT. The effect of HED was studied among cancer patients receiving immune check point inhibitors where high HED diversity was associated with better survival. Low recipient HEDs in HLA class I and DR were strong predictors for worse overall survival after matched donor HCT. The effect of HED divergence among recipients of haplo HCT with PTCY has not been evaluated. Moreover, the mismatch between donor and recipient in haplo HCT allows for assessing the effect of HED divergence in the donor and in the donor/recipient mismatched haplotype. To study the effect of HED among recipients of haplo HCT, we analyzed 322 consecutive recipient/donor pairs who received their transplant at our center with a median follow up of 57.2 months (range 18-196). HED was calculated as pairwise differences using two methods: a Grantham distance metric using a perl Script ( https://sourceforge.net/projects/granthamdist/) and manual calculation of one random sample from each analysis to verity results. Data were collected from our database where it was prospectively entered. Patient diagnosis included AML (n=122), ALL (n=68), MDS (n=41), NHL (n=50), other (n=41). Baseline recipient characteristics included a median age of 50 years (19,80), 53% white, HCT-CI≥3 in 50%, Disease risk (intermediate 53%, high/very high 33%), PBSC graft 80% and myeloablative conditioning 49%. Donor characteristics were median age 37 years (13,73), CMV positive 61%, male 61% and female donor to male recipient 21%. Pairwise divergence of HLA class I and class II showed differences in values with HLA-B, DRB1 and DQB1 contributing most to mean HED class I and II. The HED values for recipient included mean HLA class I 6.85 (HLA-A 7.08, B 8.24 and C 3.47), class II 8.58 (DRB1 10.97, DQB1 10.06 and DPB1 4.06). Donor mean HED HLA-class I was 6.5 (HLA-A 6.43, B7.93 and C 4.61), mean HLA-class II was 8.84 (DRB1 10.93, DQB1 11.24 and DPB1 4.06). The mean HEDs for the mismatched haplotype between donor and recipient were class I 6.49 (A 6.43, B 7.8, C4.94) and Class II 8.74 (DRB1 10.48, DQB1 10.06, DPB1 4.06). Next, we performed a univariate Cox model to identify HED variables that are associated with OS, DFS, NRM and relapse. Based on continuous and dichotomized divergence (median cutoff) univariate cox, a high HED in class I mismatched donor-recipient haplotype was significant for worse DFS (HR 1.11, p=0.020), and relapse (HR 1.11, p=0.02). Also, high HED in HLA B in mismatched haplotype was significant for worse OS (HR 1.07, p=0.02), DFS (HR 1.09, p=0.002), higher relapse (HR 1.10, p=0.003) and similar NRM to low HED. AS HLA-B was the biggest contributor to class I HED mean and the only predictor of outcomes in a univariate cox, we evaluated HLA-B in mismatched haplotype ( donor-recipient) further in a Multivariate analysis using dichotomization based on median level. Variables in the analysis included were age (<55, ≥55), gender, race, diagnosis, cell source, regimen intensity, HCT-CI (0-2, ≥3), DRI (low/intermediate, high/very high), CMV status; donor age (<38, ≥38), donor gender, female-donor-to-male-recipient, donor CMV status, year of transplant. The multivariate cox analysis showed that high HED (≥7.8 vs <7.8) was significant for worse DFS ( HR 1.53, 95% CI 1.09-2.16; p=0.01), higher relapse (HR 1.61, CI 1.07-2.44, p=0.024) and similar NRM and OS (table 1) . The significance was reproduced in the lymphoid and myeloid lineages. In Summary, among patients receiving haplo with PTCY, a high divergence in HLA-B mismatch haplotype increases relapse risk and worsens DFS. In addition to this being the first analysis to assess HED in a large haplo cohort, the negative consequences of HED can be mitigated by selecting donors with lower HED in the mismatched haplotype at HLA-B. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Low-Dose Antithymocyte Globulin Plus Low-Dose Post-Transplant Cyclophosphamide for Prevention of Graft-Versus-Host Disease after Haploidentical Peripheral Blood Stem Cell Transplants: A Large Sample, Long-Term Follow-up Retrospective Study

Background: The use of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) to treat hematological diseases is increasing. Two regimens for in vivo T-cell depletion are currently used in haplo-HSCT for prophylaxis of graft-versus-host disease (GVHD), including anti-thymocyte globulin (ATG)-based and posttransplant cyclophosphamide (PTCy)-based regimens. Our previous studies have demonstrated that low-dose ATG plus low-dose PTCy (low-dose ATG/PTCy) combined with cyclosporine (CSA) and mycophenolate mofetil had promising activity for the prevention of GVHD in haploidentical peripheral blood stem cell transplantation (haplo-PBSCT). Now, the efficacy of the low-dose ATG/PTCy regimen for GVHD prevention in haplo-HSCT was evaluated in a large sample with a long-term follow-up. Methods: The patients undergoing haplo-PBSCT between May 2017 and June 2021 in our center were enrolled in the retrospective study. The study included a variety of hematological diseases, such as acute myeloid leukemia (AML, n=113), acute lymphoblastic leukemia (ALL, n=49), mixed phenotype acute leukemia (MPAL, n=4), myelodysplastic syndromes (MDS, n=18), chronic myeloid leukemia (CML, n=10), non-Hodgkin's lymphoma (NHL, 30) and multiple myeloma (MM, n=1). All patients received low-dose ATG/PTCy regimen for prophylaxis of GVHD including ATG 2.5 mg/kg administered on day −2 to −1 and cyclophosphamide (Cy) 50 mg/kg on day +3, cyclosporine A (CsA) and mycophenolate mofetil (MMF) initiating on day +4. The starting infusion dose of CsA was 2 mg/kg, after which the dose was modified to obtain a nadir serum level between 200 and 300 ng/ml, eventually tapering from day +90 to day +180. MMF was administered orally at 15 mg/kg three times daily (maximum dose of 3 g per day) until day +34 and discontinued if no aGvHD. Mycophenolic acid sodium enteric tablets (MPA) correspond to MMF instead. Grafts were from mobilized peripheral blood stem cells. Family members selected as haploidentical donors were defined on the HLA-A, -B, -C, -DRB1, and -DQB1 locus at the high-resolution level of the recipient donor number of human leukocyte antigen (HLA) mismatches >2. Results: Two hundred and twenty-five patients undergoing haplo-PBSCT were enrolled in the study. There were 144 males and 81 females with a median age of 38 years (range, 6 to 69 years). At the time of transplantation, 146 (64.89%, 146/225) patients were in complete response (CR), while 79 (35.11%, 79/225) patients had active disease. One hundred and five patients received myeloablative conditioning (MAC) regimens, while 40 patients received reduced-intensity conditioning (RIC) regimens. All patients received PBSC grafts with the median CD34+ cells of 10.3 (1.64-46.55) × 108/kg, and the median mononuclear cells (MNCs) was 15.35 (3.48-39.74) × 108/kg. Ten patients failed to engraft, including 6 patients with primary graft failure and 4 with secondary graft failure. The median time for neutrophil engraftment was 12 days (range, 9-28 days), while the median time for platelet engraftment was observed in 13 days (range, 9-87 days). The cumulative incidence (CI) of grades I-IV acute GVHD (aGVHD) was 30.7% (24.6-36.9%). The CIs of grades II-IV and III-IV acute GVHD by 100 days post-transplant were 12.4% (95%CI, 8.0-16.8%) and 4.6% (95%CI, 1.8-7.4%), respectively. While chronic GVHD was 31.2% (95%CI, 24.6-37.9%) and moderate/severe chronic GVHD was 16.4% (95%CI, 11.1%-21.7%). A total of 90 patients died. Fifty-nine patients died of non-relapse causes (NRM 59/225, 26.2%). With a median follow-up of 26 months (range, 0-61 months), 50 cases relapsed. The median time of relapse was 15.5 months (range, 1−46 months). The mean OS and DFS were 38.01 months (95%CI, 34.33-41.6%) and 35.45 months (95%CI, 31.75-39.15%). The 2-year disease-free survival (DFS), overall survival (OS), and GVHD-free, relapse-free survival (GRFS) was 54.8% (95%CI, 48.94-62.26%) and 60.9% (95%CI, 54.24-67.56%) and 48.0% (95%CI, 41.14-54.86%), respectively. The 2-year CI of relapse was 26.6% and NRM was 16.6%. All patients were included in immune reconstitution studies and 81 cases were analyzed at each endpoint. On days +120, median CD3+, CD4+, CD8+, CD19+ and CD56/CD16+ counts were 954 (95-4891), 180 (8-743),754 (73-4272), 34 (1-276) and 215 (19-2539)/μl, respectively. Conclusion: The low-dose ATG/PTCy regimen presented a high efficacy for prophylaxis of GVHD in haplo-PBSCT.

Anticipation of Relapse and Acute Graft-Versus-Host Disease after Allogeneic Peripheral Blood Stem Cell Transplantation: The Fundamental Role of Antigen-Presenting (Dendritic) Cells.

BackgroundDendritic cells (DCs) are antigen-presenting cells. In humans two distinct lineages of DCs exist: DC1 and DC2. Efforts to explore the role of DCs in acute graft-versus-host disease (aGVHD) after allogeneic peripheral blood stem–cell transplantation (PBSCT) are gaining traction. However, further research is needed to identify particular lineages and their values in terms of developing an evidence-based aGVHD- or relapse-prevention strategy. We monitored DC counts and subsets in PBSC grafts while harvesting stem cells in recipients to elucidate their value in anticipating disease relapse or aGVHD.MethodsWe enrolled 29 participants. Using fluorescence-activated cell sorting, total counts/kg of CD34+, DCs, and DC subsets were analyzed in 29 PBSC-graft components using CMRF44, CD11c, and CD4 monoclonal antibodies (MoAbs).ResultsIn the 29 grafts, we detected a significant positive correlation (P<0.01) between DCs and both DC1 and DC2. Significantly higher counts (P<0.01) of DCs and DC1 in those who had developed aGVHD (nine cases) were also observed. Relapsed cases (two) were also associated with higher counts of DCs and DC2. A significant positive correlation (P<0.05), was recorded between DCs and DC1 counts and the day of myeloid engraftment, while this was not detected on the day of platelet engraftment. Myeloid engraftment transpired earlier in patients without aGVHD. Increased DC-graft numbers, particularly DC1 measured by CD11c Moabs, were associated with aGVHD. Recipients of higher numbers of CD4bright DCs had an increased risk of relapse after allogeneic PBSCT.ConclusionThis study analyzed DCs in PBSC grafts, using novel specific MoAbs and flow cytometry. Our data showed that higher donor DC1 counts were linked to the incidence of aGVHD and DC2 with relapse. We propose a fundamental role for DC-graft monitoring in anticipating aGVHD and disease relapse.

A Novel CD34-Specific T-Cell Engager Efficiently Depletes Stem Cells and Acute Myeloid Leukemia Cells in Vitro and In Vivo

ASH Abstract.IntroAcute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS) are poor prognosis hematological malignancies characterized by abnormal hematopoiesis and dysfunctions of the hematopoietic stem cell system. Chemotherapy remains the standard of care but is associated with side effects and often high rates of relapse. Today, less than a third of patients diagnosed with AML are cured.Bispecific T-cell engagers (BiTEs) are promising immunotherapeutic agents intended for cancer treatment. BiTEs are small molecules constructed of two single chain variable fragments (scFv) connected in tandem by a flexible linker that acts by retargeting T-cells against tumor cells. One scFv binds to CD3, while the second scFv binds to a tumor-associated antigen. This structure and specificity allow a BiTE construct to physically link a T-cell to a tumor cell, stimulating effector cell activation ultimately leading to cytokine production and tumor killing.MaterialBiTEs against CD34/CD3 and relevant controls were constructed by recombinant DNA technology and purified from the supernatants of transfected CHO cells following standard procedures.The scFv domain binding to CD34 is positioned N-terminally, and the scFv binding to CD3e C-terminally followed by a hexa-histidine sequence.ResultsBy co-culturing T-cells and target AML cells for 48 h in the presence of increasing concentrations of BiTE or controls, we observed that CD34-BiTE efficiently triggered T-cell-mediated depletion of the CD34 hi and CD34 low cell lines, while negative controls killed none of the target cell lines.Next, we examined the T-cell activation and proliferation. We observed that both CD4+ and CD8+ T-cells presented high levels of CD25/CD69 expressions when the CD34+ cell lines were co-cultured with T-cells in the presence of the CD34/CD3 BiTE. No unspecific activation was found when CD34- cell line was used as target cell.Since CD34 is constitutively expressed by HSCs, the CD34-specific BiTE may deplete not only CD34 +AML blasts but also healthy HSCs. To test this, T-cells and HSCs were purified from PBSC grafts and co-cultured in the presence of either CD34/CD3 BiTE or controls. After co-culture, a significant depletion of CD34 + HSCs was observed for the CD34/CD3 BiTE.To address the potential of the anti-CD34 BiTE in vivo, we next established a human CD34 + cell line in NSG mice per intravenous injection and randomized into three different groups and started treatment the day after. Two groups of mice received two consecutive cycles of one intraperitoneal injection of freshly isolated human T-cells followed by daily intravenous injections of either BiTE or control. The mice were euthanatized at day 21 by which the AML burden was measured, and T-cells quantified. No side effects of the treatment, including after BiTE administration, was observed. There were statistically significant reductions of leukemia burden in both bone marrow and spleen in mice receiving T-cells and BiTE compared to T-cells only and control.ConclusionsWe show that the CD34/CD3 BiTE is able to promote T-cell activation and killing of CD34-expressing target cells with high efficacy in vitro and in vivo, supporting the translation of this drug into clinical trials.In this scenario, the treatment with CD34-targeting BiTE prior to HSCT would trigger the patient's T-cells to deplete CD34 + leukemic blasts and HSCs. As consequence, this adjuvant treatment would decrease the use of cytotoxic and cytostatic conditioning drugs before HSCT, reducing life-threatening complications such as GvHD and infections. DisclosuresArruda: Anocca: Current Employment, Research Funding. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mattsson: MattssonAB medical: Current Employment, Current holder of individual stocks in a privately-held company. Onfelt: Desumo: Current Employment, Current holder of individual stocks in a privately-held company. Uhlin: XNK therapeutics: Current Employment, Current holder of stock options in a privately-held company.

Optimizing Donor Selection for Haploidentical Transplantation Utilizing the Four-Group T Cell Epitope (TCE-4) Algorithm for Prediction of HLA-DPB1 Non-Permissive Mismatches

▪An HLA-DPB1 non-permissive mismatch (npmm) has been associated with higher risks of acute graft-versus-host disease and non-relapse mortality after matched unrelated donor transplantation (MUDT) and thus avoiding HLA-DPB1 npmm is important in unrelated donor selection. In contrast, HLA-DPB1 npmm by the 3-group T cell epitope algorithm (TCE-3) has been shown to be protective in the context of haploidentical donor transplantation (HIDT) using post-transplant cyclophosphamide (PTCy) (Solomon et al. BBMT 2018). Additional HLA-DPB1 “permissiveness” models, also based on cross-reactive patterns of alloreactive T cells against HLA-DPB1 alleles, include the TCE-4 (based on 4 TCE groups) and functional distance (FD, based on net difference in distance between key amino acid polymorphisms) algorithms. Lastly, an expression model is based on the surface expression of the recipient mismatched HLA-DPB1 (RDP) allele according to variants of a biallelic SNP. The present analysis had 2 major aims: to 1) determine which HLA-DPB1 permissiveness model (TCE-3, TCE-4, FD, expression) provides the best tool for haploidentical donor selection and 2) analyze the role of vector (GVH vs. HVG direction) on the effect of an HLA-DPB1 npmm. A total of 322 patients with acute leukemia, MDS, lymphoma, CLL or CML, receiving a HIDT-PTCy from a single institution were evaluated with a median follow-up time of 45 months [range 6, 184]. Baseline characteristics included a median age of 50 years [19, 80], 47% non-white, HCT-CI ≥3 in 50%, PBSC graft in 80%, and myeloablative conditioning in 49%. The number of donor-recipient pairs having an HLA-DPB1 npmm according to the TCE-3, TCE-4, FD and expression was 82 (25%), 130 (40%), 54 (17%) and 99 (31%) respectively. In univariate analysis, HLA-DPB1 npmm identified by the TCE-3 and TCE-4 models were statistically associated with improved overall survival (OS) (p=0.041 and p=0.004 respectively), whereas FD risk and RDP expression were not (see figure). For disease-free survival (DFS), only the TCE-4 model showed a statistically significant association (p=0.022). Directionality of the HLA-DP npmm (GVH vs. HVG vector) had no significant impact on survival following HIDT-PTCy, a finding similar to the context of MUDT (Fleischhauer BMT 2017). In multivariate Cox analysis, adjusting for patient/donor age, gender, race, HLA-DR mismatch and transplant year, HLA-DPB1 npmm by the TCE-4 model had the most significant association with improved OS (HR 0.59, p=0.012), with TCE-3 being less predictive (HR 0.65, p=0.07) (see figure). Furthermore, HLA-DPB1 npmm by TCE-4 led to improved DFS (HR 0.69, p=0.046) and trends for lower cumulative incidences of relapse/progression (HR 0.73, p=0.16) and NRM (HR 0.54, p=0.09). In summary, the presence of an HLA-DP npmm in either the GVH or HVG direction continues to be associated with improved survival following HIDT-PTCy in a large single institution retrospective analysis with extended follow-up. Compared to the original TCE-3 model, a TCE-4-predicted HLA-DPB1 npmm is more strongly associated with overall survival. This fact, combined with the larger number of HLA-DPB1 npmm donors identified by the TCE-4 model, suggests that it may be a better selection tool for optimal haploidentical donor identification. [Display omitted] DisclosuresSolh: Partner Therapeutics: Research Funding; Jazz Pharmaceuticals: Consultancy; BMS: Consultancy; ADCT Therapeutics: Consultancy, Research Funding.

Recipient HLA-B Leader Genotype, and Its Relationship to Total KIR Missing Ligand, Informs Relapse and Survival Following Haploidentical Transplantation Using Post-Transplant Cyclophosphamide

▪In addition to donor T cells, natural killer (NK) cells are proposed to play a significant role in the graft-versus-leukemia (GVL) effect following haploidentical donor transplantation (HIDT). Following HIDT, donor NK cells express activating (NKG2C) receptors (Ciurea, Leukemia 2021), whose ligand is the non-classical human leukocyte antigen, HLA-E. For surface expression, HLA-E requires binding of leader peptides derived from class I HLA molecules. The rs1050458C/T dimorphism at position -21 of exon 1 of HLA-B gives rise to leader peptides with either methionine (M) or threonine (T) at the second residue of the processed leader peptide. M-containing HLA-B leader peptides promote higher HLA-E expression than T-leaders, potentially favoring more robust natural NK cell recognition of HLA-E-expressing tumor cells. Alternatively, donor NK cells can be activated through inhibitory killer cell immunoglobulin-like receptors (iKIR: 2DL1, 2DL23, 3DL1, 3DL2), if they fail to engage a corresponding class I HLA ligand on recipient leukemia cells (missing ligand (ML) paradigm). We hypothesized that M-containing B-leaders (either MM or MT genotype), and potentially its association with KIR ML, may inform relapse and survival after HIDT using post-transplant cyclophosphamide (PTCy). A total of 322 patients with acute leukemia, MDS, lymphoma, CLL or CML, receiving a HIDT-PTCy from a single institution were evaluated with a median follow-up time of 45 months [range 6, 184]. Baseline characteristics included a median age of 50 years [19, 80], 47% non-white, HCT-CI ≥3 in 50%, PBSC graft in 80%, and myeloablative conditioning in 49%. M-containing B-leader genotype (either MM or MT) was seen in 42% and 44% of recipients and donors, respectively. The B-leader on the unshared donor-recipient haplotypes was matched (either T or M) in 61% of transplants. ML for iKIR 2DL1, 2DL23, 3DL1, and 3DL2 was noted in 29%, 20%, 24% and 72% respectively. Total ML was 0, 1, 2 and 3 in 10%, 43%, 40% and 7% respectively. In univariate analysis, an M-containing recipient B-leader genotype [R(M+)] improved OS and DFS compared to a recipient TT genotype [R(M-)] (75 vs. 53%, p<0.001; 66 vs. 47%, p<0.001), which was primarily due to a lower risk of relapse/progression (24% vs. 38%, p=0.012). In regard to total iKIR ML, the presence of 2-3 ML was associated with better overall (OS) and disease-free (DFS) survival compared with 0-1 (67% vs. 57%, p=0.08; 62% vs. 48%, p=0.019), which was due to lower relapse/progression (25% vs. 38%, p=0.015). There was no association of either recipient B-leader or total iKIR ML with the incidence of NRM, acute or chronic GVHD. When recipient B-leader and ML were combined, the detrimental effect of a R(M-) genotype was seen exclusively in patients with ML 0-1 (see figure). DFS for R(M-)/ML(0-1), R(M+)/ML(0-1), R(M-)/ML(2-3), R(M+)/ML(2-3) was 36%, 68%, 65% and 60%, respectively (p<0.001). Corresponding relapse/progression rates were 49%, 22%, 26% and 25%, respectively (P<0.001). In multivariate analysis, controlling for patient age/sex/race, disease risk index, donor age, HLA-DR mm, HLA-DP npmm and year of transplantation, both recipient B leader and total iKIR ML were independently associated with DFS (HR 0.57, p=0.002 and HR 0.67, p=0.026) and relapse/progression (HR 0.55, p=0.006 and HR 0.55, p=0.007). In summary, a recipient M-containing B-leader genotype (MM or MT) reduces relapse and improves survival following HIDT-PTCy, presumably through HLA-E-mediated effector cell engagement. Furthermore, the negative consequences of low total ML for iKIR, in terms of increased relapse risk and lower DFS, can be mitigated when a R(M+) B-leader is present. In addition to the clinical importance of this novel finding for optimizing HIDT-PTCy, it further supports the role of alloreactive donor NK cells for optimal GVL in this context. [Display omitted] DisclosuresSolh: Partner Therapeutics: Research Funding; Jazz Pharmaceuticals: Consultancy; ADCT Therapeutics: Consultancy, Research Funding; BMS: Consultancy.

Low Incidence of Chronic Graft-Versus-Host Disease in Myeloablative Allogeneic Hematopoietic Cell Transplantation with Post-Transplant Cyclophosphamide Using Matched Related or Unrelated Donors: Phase II Study Interim Analysis

Low Incidence of Chronic Graft-Versus-Host Disease in Myeloablative Allogeneic Hematopoietic Cell Transplantation with Post-Transplant Cyclophosphamide Using Matched Related or Unrelated Donors: Phase II Study Interim Analysis

Risk Factors for the Incidence of and the Mortality due to Post-Transplant Lymphoproliferative Disorder after Hematopoietic Cell Transplantation

Post-transplant lymphoproliferative disorder (PTLD) is a potentially serious complication that occurs following hematopoietic cell transplantation (HCT), in which B cells transformed by Epstein-Barr virus (EBV) proliferate uncontrollably. It is unknown whether risk factors for the incidence of PTLD are identical to those for mortality due to PTLD, a clinically more important outcome. We sought to determine the risk factors influencing the incidence of PTLD and those influencing mortality due to PTLD in a cohort of 1184allogenic HCT recipients. All patients were predisposed to PTLD, because their graft-versus-host disease (GVHD) prophylaxis included antithymocyte globulin. The overall PTLD incidence was 9.0%, and mortality due to PTLD was1.1%. Inmultivariate analysis, risk factors for PTLD incidence include donor+/recipient- (D+/R-) EBVserostatus (subhazard ratio [SHR], 3.3; P=.002), use of a donor other than an HLA-matched sibling donor (non-MSD) (SHR, 1.7; P=.029), receipt of total body irradiation (TBI; SHR, 3.3; P=.008), and the absence of GVHD (SHR, 3.3; P < .001). The sole risk factor for mortality due to PTLD among all patients was D+/R- serostatus (SHR, 5.8; P=.022). Risk factors for mortality due to PTLD among patients who developed PTLD were use of a bone marrow (BM) graft (compared with peripheral blood stem cells [PBSCs]; SHR, 22.8; P < .001) and extralymphatic involvement (SHR, 14.6; P < .001). Interestingly, whereas the absence of GVHD was a risk factor for PTLD incidence, there was a trend toward the presence of GVHD as a risk factor for PTLD mortality (SHR, 4.2; P=.093). Likewise, whereas use of a BM graft was a risk factor for PTLD mortality, there was a trend toward use of a PBSC graft as a risk factor for PTLD incidence (SHR, 0.44; P=.179). Some risk factors for the incidence of PTLD are identical to the risk factors for mortality due to PTLD (ie, D+/R- serostatus), whereas other risk factors are disparate. Specifically, TBI was identified as a risk factor for PTLD incidence but not for PTLD mortality; the absence of GVHD was a risk factor for PTLD incidence, whereas the presence of GVHD was possibly a risk factor for PTLD mortality; and receipt of a PBSC graft was possibly a risk factor for PTLD incidence, whereas receipt of a BM graft was a risk factor for PTLD mortality.

Myeloablative Conditioning With PBSC Grafts For T-Replete Haploidentical Donor Hematopoietic Cell Transplantation Using Post-Transplant Cyclophosphamide Results In Universal Engraftment, Low Rates Of Gvhd, NRM and Excellent Survival Outcomes: An Analysis Of Two Consecutive Phase II Studies From a Single Center

Lack of a matched sibling or suitably matched unrelated donor can be a significant barrier to allogeneic hematopoietic cell transplantation in patients who stand to benefit from this procedure. HLA-haploidentical donors are readily available for nearly all such patients. Whereas previous attempts at haploidentical donor hematopoietic stem cell transplantation (haploSCT) using standard immunosuppression or ex-vivo T-cell depletion have had relatively poor outcomes, a recent approach using a T-replete bone marrow graft with non-ablative conditioning and post-transplant cyclophosphamide (ptCy) for control of alloreactivity has proved to be remarkably well tolerated (Luznik et al BBMT 2008). However, relapse of malignancy remains a major challenge with the use of this non-myeloablative regimen. The use of myeloablative conditioning and a PBSC graft may help reduce the relapse rate following T-replete haploSCT but this has not been well studied. We enrolled a total of 41 patients on two consecutive prospective phase II trials of myeloablative haploSCT and ptCy using a T-replete G-CSF mobilized PBSC graft. The first trial (NSH 864) employed a high-dose busulfan-based preparative regimen (fludarabine 30 mg/m2/d d-6 to -2, Busulfan 110-130 mg/m2/d d-7 to -3 , Cy 14.5 mg/kg/d d-6&-5) [n=20], the second (NSH 922) utilized a TBI-based regimen (fludarabine 25 mg/m2/d x 3 d and TBI 150 cGy bid on d -4 to -1 [total dose 1200cGy]) [n=21]. Post-grafting immunosuppression consisted of Cy 50mg/kg/day on days 3 and 4, MMF through d 35, and tacrolimus through d 180 on both protocols. Eligibility was limited to patients ≤60 years of age with aggressive/advanced hematologic malignancies perceived to be at high risk of relapse following nonmyeloablative transplant. Leukemia patients in CR1 were eligible if they had either poor-risk cytogenetics/molecular studies or primary chemoresistance. Median patient age was 45 years (range 24-60). Twenty-five patients were transplanted in complete remission (CR) or in chronic phase (CP), while 16 patients were transplanted with advanced disease. No patient experienced graft failure, with a median time to neutrophil and platelet recovery of 16 and 25 days, respectively. All evaluable patients achieved sustained complete donor T cell and myeloid chimerism by Day +30. Median follow-up for survivors is 20 months (NSH 864 - 39 months; NSH 922 – 11months). The cumulative incidence of grades II-IV and III-IV acute GVHD at day +100 was 30% and 17% respectively. The cumulative incidence of chronic GVHD at one year post transplant was 41% (severe in 9%). Non-relapse mortality (NRM) at 12 and 24 months was 8% and 17% respectively. Cumulative incidence of relapse was 31% at both 12 and 24 months post-transplant. The relapse rate was lower in patients transplanted in CR/CP (18%) vs. those transplanted with advanced disease (49%, p=0.044). BK virus-associated hemorrhagic cystitis (HC) occurred in 56% of patients, and was severe (requiring hospital admission for bladder irrigation and/or pain management) in 19%. The incidence of HC and severe HC was significantly lower in patients receiving TBI-containing conditioning than those receiving Busulfan (38% vs. 75%, p=0.028 and 10% vs. 30%, p=0.13, respectively). With a median follow-up of 20 months, the estimated 2 year overall survival (OS) was 51% with a 52% estimated disease-free survival (DFS). In patients transplanted in CR/CP, 2-year DFS was 64% vs. 35% in patients transplanted with advanced disease (p=0.077). There was an encouraging trend to better 2-year DFS in patients receiving TBI-based conditioning than those receiving Busulfan, 75% vs. 40%, p=0.072. Myeloablative haploSCT with PBSC grafts and ptCy allows reliable engraftment with early donor T cell chimerism; low rates of GVHD, infectious complications, and NRM; and promising survival in patients with high-risk hematologic malignancies. Outcomes are particularly good in patients treated in CR/CP (18% relapse, 64% DFS). When compared with Busulfan-based conditioning, TBI-based conditioning is associated with less risk of HC and improved DFS. Myeloablative haploSCT is a valid option for patients with aggressive/advanced hematologic malignancies who lack timely access to a conventional donor.▪▪ Disclosures:No relevant conflicts of interest to declare.

Individual Patient Dose-Escalated Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-Vs.-Host Disease in Children and Adults: Safety, Efficacy and Immune Correlates

CD4+CD25+FoxP3+ regulatory T cells (Treg) are broadly suppressive and play a central role in the prevention of chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic cell transplantation (HCT). Daily subcutaneous (SC) low-dose interleukin-2 (IL-2) at a fixed dose of 1x106 IU/m2/day in adult patients with refractory cGVHD induces selective Treg expansion and leads to clinical responses in 50-60% of evaluable participants (NEJM 2011, Blood 2016). During daily fixed-dose IL-2 therapy, plasma IL-2 levels rose rapidly by week 1, but subsequently decreased over time. The lower IL-2 levels were associated with a decline in Treg proliferation despite daily IL-2 administration (Sci Transl Med 2013), possibly due to IL-2 sequestration via binding to constitutively-expressed high affinity IL-2 receptors on Treg. We reasoned that IL-2 dose escalation at the time of anticipated fall in plasma IL-2 levels would avoid tachyphylaxis and maximize IL-2 induced Treg numbers in vivo.We conducted a phase 1 trial of individual patient IL-2 dose escalation over 8 weeks in adult and pediatric patients with steroid-refractory cGVHD. Daily SC IL-2 was initiated at 0.67x106 and 0.33x106 IU/m2/day in the adult and pediatric cohorts, respectively. Each participant had dose escalations at weeks 2 and 4 to a maximum dose of 2x106 IU/m2/day in adults and 1x106 IU/m2/day in children. We studied 18 HCT recipients (10 adult: 100% HLA-matched, 100% PBSC grafts, 40% prior aGVHD; 8 pediatric: 87.5% HLA-matched, 87.5% BM grafts, 28.6% prior aGVHD). Median age was 57 years (range, 33-71) in the adult cohort and 12 years (range, 5-22) in the pediatric cohort. The median time from HCT and from cGVHD onset to start of IL-2 treatment was 3 years (range, 1-12.1) and 597 days (range, 234-4336) respectively. Participants had a median of 2 cGVHD organ sites (range, 1-5) and 3 prior cGVHD therapies (range, 1-4) at enrollment. The cohorts did not differ in terms of time since cGVHD onset and number of failed therapies prior to enrollment.Individually dose-escalated IL-2 was well tolerated in children with 1 participant requiring dose reduction for electrolyte imbalances. In the adult cohort, 1 participant withdrew due to cGVHD progression, 1 required dose reduction for injection site reactions and 2 were unevaluable (1 IL-2 hold >1 month, 1 non-compliance). Seven grade 3 AEs were reported for six participants (electrolyte imbalance, vomiting, infection, increased ALT and soft tissue neoplasm); none relapsed. At week 8, objective cGVHD responses (PR) were seen in 7 of 8 pediatric participants (88%), but in only 1 of 8 evaluable adult participants (13%). cGVHD response sites included skin (n=4), joint/fascia/muscle (n=1), lung (n=4), and GI tract (n=2). Eight pediatric and 4 adult participants with clinical benefit (PR or SD with minor response) continued extended duration IL-2 therapy up to 120 weeks.Plasma IL-2 levels were higher in adults at weeks 1 and 2, but immune responses were similar between the 2 cohorts. Dose escalated IL-2 induced a >10-fold increase in median Treg count/µL that peaked at week 6. There were no significant changes in CD4 Tcon, CD8 T cell or B cell count. NK cell count increased >5-fold by week 8. The median Treg:Tcon ratio increased 6-fold from a baseline of 0.05 (Q1-Q3, 0.024-0.114) to a week 5 peak of 0.312 (Q1-Q3, 0.203-0.37). Compared with fixed daily dosing of IL-2 (Blood 2016), the individually dose escalated IL-2 schedule had a slower initial rise in Treg number and Treg:Tcon ratio (Figure 1). This likely reflects the lower starting doses in this study. The maximum IL-2 dose in the adults was twice the previously defined MTD but did not improve Treg expansion. Despite lower IL-2 doses, pediatric patients had a superior clinical response and a trend toward a better immunologic response compared with the adults.We demonstrate for the first time that low-dose IL-2 is safe in children with advanced steroid-refractory cGVHD and results in a high clinical response rate. A maximum dose of 1x106 IU/m2/day was well tolerated by pediatric patients in the extended duration phase and durably maintained Treg numbers above baseline. Five pediatric patients that continued IL-2 therapy for >20 weeks have reduced or discontinued steroid therapy. In adults, intrapatient dose escalation above fixed-dose MTD was not clinically tolerated and did not result in greater Treg expansion or clinical improvement relative to fixed daily dosing. [Display omitted] DisclosuresNikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Sequenta/Adaptive: Research Funding; Affimed: Research Funding; Roche: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Infinity: Consultancy; Genmab: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Otsuka: Research Funding; Pfizer: Consultancy, Research Funding; Tensha: Research Funding; Sigma Tau: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Koreth:Prometheus Labs: Research Funding; Amgen, Inc: Consultancy.

Extra-Corporeal Photopheresis Plus Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-Vs.-Host Disease: Efficacy, Safety and Immune Correlates

CD4+CD25+FOXP3+ regulatory T cells (Treg) are dominant suppressors of innate and adaptive immune effector cells that enhance tolerance. Treg augmentation can control immune activation underlying chronic graft-versus-host disease (cGVHD). In prior studies, daily subcutaneous (SC) low-dose interleukin-2 (IL-2) therapy for 8-12 weeks induced preferential Treg expansion in-vivo, with objective clinical responses in over half of evaluable participants (NEJM 2011, Blood 2016). Alternative immunomodulatory approaches like extra-corporeal photopheresis (ECP) also have efficacy in cGVHD and may function via Treg augmentation. Combining ECP plus IL-2 could offer therapeutic synergy.We report on a prospective single arm phase 2 trial of twice-weekly ECP (weeks 1-16) plus sequential low-dose IL-2 (1x106 IU/m2/d SC, weeks 9-16) for treatment of steroid-refractory cGVHD. The study enrolled 25 participants (48% male, 88% HLA-matched, 100% PBSC grafts, 52% prior aGVHD). Median participant age was 62 years (range, 34-76). Median time from HSCT and from cGVHD onset to start of treatment was 919 days (range, 177-2842) and 524 days (range, 58-2697) respectively. Participants had a median of 3 cGVHD organ sites (range, 2-5). The median follow-up time in survivors is 16 months (range, 2.1-26).ECP plus IL-2 was well tolerated: 1 participant withdrew due to IL-2 adverse effects (AE) (myalgia, fatigue), and 3 were additionally unevaluable (1 cardiac arrest; 1 parainfluenza infection, 1 still undergoing treatment). None required IL-2 dose reduction. There were four grade 3 AEs during the 16-week study (1 participant had dehydration/hypotension/syncope, possibly IL-2-related;1 had ECP-related anemia). None experienced relapse. At week 16, cGVHD objective partial responses (PR) were documented in 14 of 21 evaluable participants (67%). None had cGVHD progression. Response sites included skin (n=9/18), joint/fascia/muscle (n=7/16), liver (n=1/1), lung (n=2/9), and GI tract (n=3/5). 18 participants with clinical benefit (PR, and stable disease with minor response) proceeded to extended ECP plus IL-2 therapy.Immunologically, 8 weeks of ECP alone did not induce significant changes in buffy coat WBC or absolute lymphocyte count (ALC). Peripheral blood CD3 T cell counts/µL fell, with a median CD8 and CD4 conventional T cell (Tcon) decrement of -109 (Q1, Q3: -220, -13; p<0.001) and -68 (Q1, Q3: -244, -19; p=0.023), respectively, at week 8 (Figure 1A). There was an early fall in Treg cell counts/µL, with a median decrement of -4 (Q1, Q3: -9, 0; p=0.033) at week 4, without change in NK or B cell counts or Treg:Tcon ratio. The addition of low-dose IL-2 thereafter induced a >5-fold increase in median Treg count/µL (p<0.001), from a baseline of 9 (Q1, Q3: 5, 17) at week 8 to a peak of 72 (Q1, Q3: 22, 186) at week 10, plateauing at 50 (Q1, Q3: 33, 71) at week 16 (Figure 1A). No significant change in CD4 Tcon or B cell count was noted. NK cell count increased 1.7-fold (p=0.009). The median Treg:Tcon ratio increased 4-fold (p<0.001): from a baseline of 0.06 (Q1, Q3: 0.03, 0.08) at week 8 to a peak of 0.26 (Q1, Q3: 0.12, 0.42) at week 12, and remained elevated at 0.17 (Q1, Q3: 0.14, 0.34) at week 16 (Figure 1B). Treg count and Treg:Tcon ratio remained elevated in responders during extended ECP plus IL-2 therapy.Immunologically, clinical responders had a higher median Treg count/µL vs. non-responders after 1 and 6 weeks of IL-2 therapy (21 vs. 11, p<0.001; 73 vs. 29, p=0.016 respectively). Treg:Tcon ratio at week 2 and 6 after IL-2 therapy was also higher in responders (0.29 vs. 0.15, p=0.038; 0.28 vs. 0.13, p=0.016 respectively). Applying our previous cutoffs (Blood 2016), Treg:Tcon ratio ≥0.2 after 1 week of IL-2 was also predictive of clinical response (p=0.05).In summary, in steroid-refractory cGVHD, ECP was associated with a fall in peripheral CD8, CD4 Tcon, and Treg counts. The addition of low-dose IL-2 thereafter induced profound Treg and NK cell augmentation. Clinical responses (PR) to combination ECP plus IL-2 therapy were documented in 67% of the evaluable participants. Those with early rise in Treg count and Treg:Tcon ratio after the addition of IL-2 were likelier to benefit. However, the overall clinical efficacy of ECP plus IL-2 did not appear markedly higher than low-dose IL-2 alone. [Display omitted] DisclosuresKoreth:Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy. Armand:Bristol-Myers Squibb: Consultancy, Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Sequenta/Adaptive: Research Funding; Sigma Tau: Research Funding; Tensha: Research Funding; Affimed: Research Funding; Roche: Research Funding; Pfizer: Consultancy, Research Funding; Genmab: Consultancy; Infinity: Consultancy; Otsuka: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

A prospective single-center study on CNI-free GVHD prophylaxis with everolimus plus mycophenolate mofetil in allogeneic HCT

We report a single-center phase I/II trial exploring the combination of everolimus (EVE) and mycophenolate mofetil (MMF) as calcineurin inhibitor (CNI)-free GVHD prophylaxis for 24 patients with hematologic malignancies and indication for allogeneic HCT after a high dose or reduced-intensity ablative conditioning. The study was registered as EudraCT-2007-001892-12 and Clinicaltrials.gov as NCT00856505. All patients received PBSC grafts and no graft failure occurred. 7/24 patients (29%) developed acute grades III and IV GVHD (aGVHD), 16/19 evaluable patients (84%) developed chronic GVHD (cGVHD) of all grades, and 6/19 (31.6%) of higher grades. No severe toxicities related to study medication were observed. The median follow-up of all surviving patients is 2177 days. The 3-year OS was 45.2% (95% CI: 27.4–61.4%), and the 3-year PFS was 38.7% (95% CI: 22.0–55.1%). The cumulative incidence of relapse at 1 year and 3 year was 25% (95% CI: 12.5–50.0%), and 33.3% (95% CI: 18.9–58.7%), the cumulative incidence of NRM at 1 year and 3 years was 20.8% (95%CI: 9.6–45.5%), and 29.2% (95%CI: 15.6–54.4%), respectively. The utilization of CNI-free GVHD prophylaxis with EVE+MMF resulted in high rates of acute and chronic GVHD. Therefore, we do not recommend a CNI-free combination of mTOR inhibitor EVE with MMF as the sole GVHD prophylaxis. In subsequent studies, this combination should be modified, e.g., with further components like post-transplant cyclophosphamide (PTCy) or anti-thymocyte globulin (ATG).

Secondary Graft-Versus-Host Disease (GVHD) Prophylaxis with Oral Proteasome Inhibitor Ixazomib Is Associated with Low Incidence of Recurrent, Late Acute and Chronic GVHD and Facilitated Calcineurin Inhibitor Taper within the First Year Post Allogeneic Stem Cell Transplantation

Secondary Graft-Versus-Host Disease (GVHD) Prophylaxis with Oral Proteasome Inhibitor Ixazomib Is Associated with Low Incidence of Recurrent, Late Acute and Chronic GVHD and Facilitated Calcineurin Inhibitor Taper within the First Year Post Allogeneic Stem Cell Transplantation