Neutrophil Recovery Research Articles

Real-Life Efficacy and Safety of Vemurafenib Plus Rituximab (V+R) in Relapsed or Refractory Hairy Cell Leukemia: A Multi-Center Retrospective Study (HCL-PG03R)

BACKGROUND Hairy cell leukemia (HCL) is a BRAFV600E-mutated indolent B-cell neoplasm (Tiacci et al., NEJM 2011) often relapsing after chemotherapy with purine analogs. BRAF inhibition is very active in relapsed/refractory (R/R) HCL, but produces mostly partial remissions (PR) and never clears minimal residual disease (MRD) (Tiacci, Park, et al., NEJM 2015). In a recent phase-2 single-center study on 30 R/R patients (pts) with a median of 3 prior therapies, a short chemotherapy-free regimen of the BRAF inhibitor vemurafenib (V) + rituximab (R; Mabthera) led to 87% complete remissions (CR), 60% MRD-negativity and 85% relapse-free survival (RFS) at a median follow-up of 34 months, with a favorable toxicity profile (Tiacci et al., NEJM 2021). METHODS A multi-center retrospective study was conducted to validate in a real-world setting the off-label use of V+R in R/R HCL needing therapy due to cytopenia(s). RESULTS From 6/2019 to 12/2021, 35 pts (median age 56 years, range 42-80) were treated at 14 Italian centers with VR (V: 960 mg bid for 8 weeks; R: 375 mg/m2 every 2 weeks for 8 doses); biosimilar R was used in 89% of pts. Prior therapies (median 2; range 0-11) included chemotherapy with cladribine or pentostatin (94% of pts), interferon-alpha (29%), rituximab (31%), splenectomy (6%), BRAF inhibitor monotherapy (6%) and zanubrutinib (3%). Median blood counts across the 35 pts were: platelets 69000/mmc, neutrophils 860/mmc and hemoglobin 11.6 g/dl (with 20% of pts, i.e. 7/35, requiring transfusions). Eleven pts (31%) had active (n=3) or latent (n=8) infections, including: ongoing systemic atypical mycobacteriosis (n=1); ongoing miliary tuberculosis (n=1; a 80-year old pt who was the only one receiving V+R as first-line therapy); ongoing large cerebral abscess (n=1); HIV infection controlled by anti-viral therapy (n=1); and latent tuberculosis (n=1) or HBV (n=6) infections requiring anti-microbial prophylaxis. Toxicity was as expected, mostly of grade 1-2 and always reversible (Table), allowing high relative dose intensities (RDI) for both drugs (V, median 97%; interquartile range/IQR 73-100%; R, 100% in 85% of pts). Similarly, among the 33/35 (94%) pts who received V for a substantial duration (≥35 days), and were thus evaluable for dose density, 20 pts (61%) completed V without ever suspending the drug, including 14/33 pts (42%) without any dose reduction. Vemurafenib toxicities were mainly represented by cutaneous rash, asymptomatic liver and pancreatic laboratory abnormalities, arthralgia, warts and photosensitivity. Three pts (9%) were not evaluable for response as they died prematurely due to atypical mycobacteriosis, complications of neurosurgery for cerebral abscess and sudden death unrelated to study drugs. Recovery of platelets (≥100000/mmc), neutrophils (≥1500/mmc) and hemoglobin (≥ 11 g/dl) occurred quickly, after a median of 2, 4 and 7 weeks after starting treatment, respectively. A CR was observed in 30/32 evaluable cases (94%; or 86%, 30/35 pts, by intention to treat/ITT). CR was obtained in all evaluable cases refractory to chemotherapy (n=8) or rituximab (n=3), or previously splenectomized (n=2) or unable to reach CR with a prior BRAF inhibitor (n=2). The only 2 evaluable pts not achieving a CR (1 PR; 1 short-lived resolution of cytopenias) received V at only 16%/50% RDI and R at only 13%/34% RDI due to drug toxicities. Analysis of MRD by allele-specific PCR for BRAF-V600E in the bone marrow aspirate was performed in 30 pts and showed MRD clearance (<0.05% mutant alleles) at a high rate: 79% (22/28 CR pts; or 65%, 22/35 pts, by ITT), which raised to 82% (66% by ITT) when including 1 additional CR case untested by PCR which had negative bone marrow flow cytometry. At a median follow-up of 21 months after the end of treatment, RFS was high among the 31 responding pts, with just 1 relapse (3%) in the only case obtaining a PR post-therapy (Figure). CONCLUSIONS This study validates in a real-life context the high efficacy and tolerability of the V+R chemo-free regimen delivered to R/R HCL pts, including the cost-saving use of biosimilar rituximab.

Evaluation of the Safety and Efficacy for Oral Mucositis Prevention of MIT-001 in Auto HSCT

Background: Oral mucositis (OM) is one of the most prevalent complications leading to severe pain, difficulty eating, prolonged hospitalization, and life-threatening septic condition in patients receiving chemotherapy containing conditioning regimen prior to autologous hematopoietic stem cell transplantation (auto-HSCT). MIT-001 is a novel small molecule functioning as a mitochondria-targeted radical trapping agent (RTA) that effectively inhibits cellular ferroptosis/necrosis and cytokine production via blockade of reactive oxygen species (ROS) generation and calcium accumulation in the mitochondria. The current clinical study was designed to evaluate the safety, efficacy by prevention of OM and pharmacokinetics of MIT-001 in patients with lymphoma or multiple myeloma (MM) undergoing chemotherapy prior to auto-HSCT. Methods: This phase IIa study consists of two parts: part 1 is an open label dose-escalating study dosing from 5 mg, 10 mg and up to 20 mg of MIT-001 to determine the recommended Part 2 Dose (RP2D) via evaluating the efficacy of OM, safety and pharmacokinetic characteristics of MIT-001, whereas Part 2 is a placebo-controlled, double-blinded, and randomized study to find the optimal dose of MIT-001 regarding the efficacy of OM and safety for the next IIb/III clinical trials. In Part 1, the total screening period was 28 days: conditioning chemotherapy/MIT-001 treatment for four to nine days followed by auto-HSCT and recovery for 14 days and discharge at 28 days following HSCT. MIT-001 was administered intravenously for 30 minutes once daily before conditioning chemotherapy for four to nine days, depending on the conditioning regimens including high-dose melphalan and BMT (intravenous busulfan, melphalan, thiotepa) for auto-HSCT. Pharmacokinetic blood samples of MIT-001 was collected at 5 time points including before administration to 24 hours after the start of the first and last administration of MIT-001. WHO criteria and NCI-CTCAE (v5.0) for OM grading were used for the severity of OM, which was evaluated daily during hospitalization and twice a week or once a week after discharge by investigator's choice. Pain related to OM was collected from patients using the Oral Mucositis Daily Questionnaire. Results: In part 1, total of sixteen patients were enrolled in which 5 patients were assigned in 5 mg and 20 mg group each, and six patients were assigned in the 10 mg group. Twelve lymphoma patients and four MM patients were pre-conditioned by BMT and Melphalan regimen, respectively. Severe OM (grade 3) were observed in two lymphoma patients treated with 10 mg and 20 mg MIT-001 (FAS: 13.3%, 2/16). Two patients with grade 3 OM had experienced radiation therapy of head and neck area within 1 year. Severe OM (> grade 3) did not occur in the four MM patients, while grade 2 was observed in two patients. None of the patients received total parental nutrition feeding due to OM. The median duration of grade 3 OM was 6 days (range, 3 to 9) and mean onset was 6 days (SD±1) after conditioning initiation. The duration and incidence of OM were not significantly different among the 3 groups. Mean area-under-the-curve (AUC) score for patient-reported soreness of the mouth and throat was not proportional to the dose of MIT-001. Time to engraftment was observed at a median time of 13 days (range, 11~ 26 days) determined by the neutrophil and platelet recovery time following auto-HSCT, which was within the normal range of period of auto-HSCT when compared to historical control. With the statistical analysis using the Kruskal-Wallis test, MIT-001 showed good PK profile with dose-proportionality in the range of 5 mg up to 20 mg. Two doses of 5 mg and 20 mg were determined as RP2D under steering committee. Serious adverse drug reactions were not reported. Part 2 is currently ongoing to evaluate efficacy of OM and safety of MIT-001 in forty-five patients with lymphoma and MM, where fifteen patients were allocated in 5 mg, 20 mg, and placebo group. Conclusions: The phase IIa part 1 results strongly suggest the therapeutic potential of MIT-001 to prevent severe OM in the patients with lymphoma and MM undergoing melphalan-containing conditioning followed by auto-HSCT.

ABO Incompatibility Haploidentical Peripheral Blood Stem Cell Transplantation Combined with a Single Cord Blood Unit for Severe Aplastic Anemia Patients

Background: For young severe aplastic anemia (SAA) patients without HLA matched donors, haploidentical hematopoietic stem cell transplantation can be used as an alternative selection. The grafts of classic Beijing protocol in China is a combination of bone marrow and peripheral blood stem cell. For patients with major or minor blood type incompatibility, bone marrow stem cells need to undergo red blood cell or plasma removal treatment. There was rare reports about haploidentical peripheral blood stem cell combined with a single unrelated cord blood unit for ABO incompatibility haploidentical stem cell transplantation in SAA patients. Objective: To observe the efficacy of ABO incompatibility haploidentcial peripheral blood hematopoietic stem cell transplantation combined with a single unrelated cord blood unit for SAA patients. Method s : Fifty-five SAA patients underwent stem cell transplantation from August 2018 to February 2022 in the First Affiliated Hospital of Xi'an Jiaotong Univeristy were divided into two groups based on donor-recipient ABO compatibility, the donor-recipient ABO matched group（BM+PB group）using haploidentical bone marrow and peripheral blood stem cell as grafts, and donor-recipient ABO mismatched group（CB+PB group）, using unrelated cord blood and haploidentical peripheral blood stem cell as grafts. The differences of hematopoietic reconstitution, acute and chronic GVHD, CMV and EB virus infection, PFS, and OS between the two groups were compared. Result s : There were 30 cases in CB+PB group and 27 cases in the BM+PB group. Except for differences in donor-recipient ABO compatibility, the other characteristics of the two groups of patients were comparable((P>0.05). One patient in the BM+PB group had primary graft failure, while the rest of the patients were successfully implanted. There were no significant statistical differences in neutrophil,platelet and red blood cells recovery rates, the incidence of acute and chronic GVHD, CMV, EB virus infection, and PTLD between the two groups of patients(P>0.05). There was no significant statistical difference in OS between the two groups of patients(P>0.05)(Figure 1). Conclusion: For SAA patients with donor-recipient ABO incompatibility could use haploidentical peripheral blood cell combined with unrelated cord blood for transplantation which had compared outcome with haploidentcal bone marrow and peripheral blood cell transplantation.

A Pilot Study to Determine the Safety and Efficacy of Repeated Hyperbaric Oxygen Therapy in Multiple Myeloma Patients Undergoing High-Dose Therapy and Autologous Stem/Progenitor Cell Transplantation

Background Serum erythropoietin (EPO) negatively impacts hematopoietic stem cell homing and engraftment. Hyperbaric oxygen (HBO) therapy can reduce serum EPO in healthy volunteers and when given prior to stem cell infusion may improve engraftment after hematopoietic stem cell transplant (HSCT). We previously performed a phase II trial randomizing patients with multiple myeloma undergoing high dose melphalan and autologous HSCT 1:1 between a single HBO treatment and no HBO. HBO was given as 100% oxygen, 2.5 ATA for a total of 90 min, in a single treatment on Day 0 prior to the transplant. A total of 99 patients were enrolled with 52 randomized to HBO. HBO significantly reduced EPO levels for the first 24 hours after auto transplant, but EPO levels quickly rebounded by 48 hours after transplant. We hypothesized multiple HBO treatments will keep EPO levels low during the duration of stem cell homing (i.e: the first 72 hours after HSCT) and may improve neutrophil and NK cell recovery. Methods We performed a pilot study to evaluate the safety and tolerability of multiple HBO treatments during autologous HSCT. Patients with multiple myeloma undergoing autologous HSCT were eligible. Patients received HBO therapy, consisting of hyperbaric oxygen at 2.5 ATA for a total of 90 minutes breathing 100% oxygen on days 0 (prior to HSCT), 1, and 2 of transplant. The primary objective of this pilot study was to determine the safety of repeated HBO therapy in the setting, as defined by treatment limiting toxicity (TLT) including occurrence of seizure disorder, pneumothorax, death, or any irreversible grade Ill or any grade IV toxicity within 24 hours of HBO therapy. Secondary objectives were to determine the effects of multiple HBO therapy on neutrophil count recovery and the effect of multiple HBO on serum EPO. Results 10 patients have been enrolled that are evaluable for the primary objective. All patients had multiple myeloma and 4 were female. 9/10 patients successfully completed all 3 HBO treatments and 1/10 completed HBO on days 1 and 2 only because of ear pain on day 0. No patients experienced treatment limiting toxicity. The median times to neutrophil and platelet engraftment was 12 days (range 11-14) and 16 days (range 14-28), respectively. The median length of hospitalization was 13 days (range 12-16). Febrile neutropenia occurred in 3/10 patients with just one incidence of documented infection. This was significantly less than the rate of febrile neutropenia in patients receiving a single treatment of HBO or no HBO (81% and 47%, respectively, p<0.05). 3 patients required red blood cell transfusion and 5 platelet transfusion. Conclusions Multiple HBO treatment were well tolerated without treatment limiting toxicity during autologous HSCT for multiple myeloma.

TCRαβ +/CD19 + - Depleted HLA-Haploidentical Hematopoietic Stem Cell Traansplantation in Pediatric Patient with Fanconi Anemia

Background Allogeneic hematopoietic stem cell transplantation (HSCT) currently represents the only proven treatment able to rescue bone marrow failure and prevent the occurrence of clonal evolution in patients with Fanconi Anemia (FA). If HSCT from HLA-identical related donor (MRD) is reported to be associated with better outcomes, this is not available for most patients. On the other hand, the identification of a fully matched unrelated donor (MUD) may be unsuccessful, or require times incompatible with a prompt treatment of the haematological condition. For patients who lack a MRD or MUD, the preferred donor choice is debated. HLA partially matched haploidentical donor (haplo) is virtually immediately available for all patients and haplo-HSCT has been reported over the last few years as a promising alternative for those patients. Following the favorable results already reported from our group of a cohort of pediatric and young adult FA patients underwent haplo-HSCT, based on the selective depletion of TCRαβ + and CD19 + cells graft manipulation, here we present the outcome of an enlarged cohort of patients who received HSCT in 3 different centers. Methods Patients diagnosed with FA and eligible for an allogeneic transplantation, but lacking a MRD or MUD, and with a suitable HLA-haploidentical donor were eligible to this study. The conditioning regimen included intravenous fludarabine, cyclophosphamide and single-dose total body irradiation for most patients. Pretransplant anti-T-lymphocyte globulin was administered in all patient to control in vivo bidirectional donor-recipient alloreactivity. Apheresis and graft manipulation were performed as previously reported (Li Pira, Biol Blood Marrow Transplant 2016). Results Thirty-five patients (19 males and 16 females) underwent haplo-HSCT between September 2011 and April 2023 in 3 different centers (Bambino Gesù Children's Hospital, Rome, Italy; Policlinico San Matteo, Pavia, Italy; Tokyo; National Center for Child Health and Development, Children's Cancer Center, Tokyo, Japan). Twenty-four patients have been previously reported (Strocchio, Blood Adv 2021). Details on patients and donor characteristics, graft composition and conditioning regimen are listed in table 1. One patient with clonal evolution and 1 with AML received fludarabine and AraC 3 weeks before the start of the conditioning. Primary engraftment was achieved in 33 patients (94.3%), with median time for neutrophil and platelet recovery of 12 days (range, 9-20 days) and 10 days (range, 7-14 days), respectively. Primary graft failure occurred in 2 patients and both were successfully transplanted from the other haploidentical parent. The monitoring of chimerism analysis confirmed a stable engraftment of donor hemopoiesis in all engrafted patients. Within 33 evaluable patients, 9 (27.3%) experienced grade 1-2 aGvHD (7 with stage I-II skin-only involvement, 1 with stage I lower gastrointestinal involvement) at a median time of 35.5 days (range, 16-67 days) after HSCT. One patient developed late-onset skin only grade 2 aGvHD at day +211 after HSCT. One patient (3%) experienced grade IV aGvHD (liver and gastrointestinal involvement) at day + 100 from HSCT, 4 weeks after the infusion of adenoviral-specific donor T cells; he is currently receiving ruxolitinib and the complete resolution of GvHD was obtained. Only one patient (3%), developed cGvHD with mild skin involvement. Thirteen patients (39.4%) experienced one or more viral infections or reactivations (6 cytomegalovirus, 4 herpesvirus-6, 2 adenovirus, 1 parainfluenzae); the specific antiviral treatment was administered with a complete resolution of infection in all cases. With a median follow up of 5.5 years (range, 0.6-11.4 years) all the patients are alive and with resolution of hematological complications. Except for the already reported vulvar intraepithelial neoplasia, no additional posttransplant malignancy was observed Conclusions Our data obtained from enlarged cohort of patients previously reported, confirm excellent results in terms of survival and a good rate of engraftment also in a multicenter setting. The low incidence of acute and chronic GvHD, a particularly detrimental complication in patients with DNA repair disorder, makes this approach attractive in this patient population. In FA patients lacking a standard donor, TCRab +/CD19 + haplo-HSCT is confirmed to be a promising strategy.

Addition of Single Dose Gemtuzumab Ozogamicin to Intensive Induction Chemotherapy in Core-Binding Factor Acute Myeloid Leukemia

Background: In a meta-analysis of 5 trials, the addition of gemtuzumab ozogamicin (GO) to intensive induction chemotherapy led to a survival benefit in patients with core-binding factor (CBF) acute myeloid leukemia (AML). The survival benefit was observed with a lower dose or fractionated schedule of GO. Given the heterogenous incorporation of GO in clinical trials, the ideal dose and schedule remains unclear. We report our outcomes of adding a single-dose of GO to intensive induction chemotherapy for patients with CBF-AML. Methods: We conducted a retrospective analysis to compare outcomes of patients with CBF-AML treated with intensive induction chemotherapy, with and without GO, at the University of Alabama at Birmingham (UAB) between January 2010 and April 2023. Single-dose GO was given at 3mg/m 2 during induction only. Results: There were 87 patients included in the analysis (GO=32, control=55). The baseline characteristics are shown in Table 1. The median age of the GO and control groups was 50y (19-68y) and 47y (21-74y), respectively. There was an overrepresentation of white patients in the control group. In the GO group, more patients had t(8;21) (53% vs. 31%, p=0.02), KIT (28% vs. 12%, p<0.01), FLT3-ITD (16% vs. 7%, p=0.03) and NRAS/KRAS mutations (40% vs. 20%, p<0.01) compared to the control group. Induction chemotherapy consisted of 7+3 in 88% of patients receiving GO and all patients in the control group. Patients <65y received idarubicin 12mg/m 2 while older patients received daunorubicin 45mg/m 2. The composite complete remission (cCR) rate was higher in the control group (93%) compared to the GO group (82%) (p<0.001). The rate of measurable residual disease (MRD) negative cCR, by flow cytometry, was similar between both groups (GO=62%, control=69%, p=0.5). There were no significant differences between the two groups in terms of time to neutrophil and platelet recovery, veno-occlusive disease, febrile neutropenia and length of hospital stay. GO was not administered during consolidation chemotherapy. The 3-year relapse-free survival (RFS) for both groups was similar (71% vs 68%, p=0.5). The 3-year overall survival (OS) for the GO group was 68%, compared to 66% for the control group (p=0.9) (Figure 1). Conclusion: In our report, we find that survival of patients with CBF-AML is favorable in the real-world setting. These outcomes are comparable to those reported in the meta-analysis leading to the approval of GO. The addition of single-dose GO did not lead to a higher remission rate or survival benefit, when compared to intensive chemotherapy without GO. However, the analysis is limited by differences in baseline characteristics between the two groups with more patients in the GO group harboring high-risk features such as the presence of t(8;21), KIT, FLT3-ITD and KRAS/NRAS mutations. Further investigation into the incorporation of GO in the treatment algorithm for CBF-AML is needed.

A Phase 2 Study of Lenalidomide in Combination with Mitoxantrone, Etoposide, and Cytarabine (Len plus MEC) in Patients with Relapsed or Refractory Acute Myeloid Leukemia

Background: Outcomes for patients with relapsed or refractory acute myeloid leukemia (R/R AML) remain extremely poor. Response rates with salvage chemotherapy regimens, such as mitoxantrone, etoposide, and Cytarabine (MEC), have been associated with a complete remission rate of around 20-30%. We previously reported a phase I study combining Lenalidomide (Len) with MEC salvage chemotherapy, with encouraging response rates in R/R AML compared to other historical standards (DeAngelo et al. AJH 2018). This led to the current phase 2 study (NCT03118466) of Len combined with MEC in R/R AML to evaluate for the efficacy of this combination. Methods: We conducted a phase 2 study of Len plus MEC in adult patients (≥ 18 yrs) with R/R AML after 1 or more cycles of induction chemotherapy. Patients received Len dosed at 50mg/day on days 1-10, and Mitoxantrone 8mg/m2/d, Etoposide 100mg/m2/d and Cytarabine 1000mg/m2/d on days 4 through 8 of induction (Kohrt et al AJH 2010). Patients who achieved a complete remission (CR), complete remission with incomplete blood count recovery (CRi) or incomplete platelet recovery (CRp) could proceed with consolidation chemotherapy or hematopoietic stem cell transplantation at the discretion of the treating physician. The study followed a Simon two-stage design powered to detect improvement to a 45% response rate. The primary objective was to determine the response rate. Secondary objectives included time to neutrophil and platelet count recovery, treatment related mortality, rates of relapse, and overall survival. Results: A total of 40 patients were enrolled and received study treatment. The median age was 56 years (range, 19-70); 58% of the patients were male. 73% of patients had relapsed after initial therapy (29/40) while 27% had primary refractory AML (11/40) after their initial induction chemotherapy. The median number of prior therapies was 2 (range, 1-4). The majority of patients had adverse ELN 2023 risk (n=22; 55%). Recurrent gene mutations in 5 or more patients included DNMT3A (n=8), TET2 (n=7), NRAS (n=6), IDH2 (n=5), and ASXL1 (n=5); 2 patients harbored TP53 mutations. The most frequent grade 3-4 treatment-related toxicities were febrile neutropenia (n = 30; 75%), platelet count decrease (n = 40; 100%), anemia (n = 18; 45%), neutrophil count decrease (n = 14; 34%), blood bilirubin increase (n=6, 14%), and anorexia (n = 5; 12%). Three patients (8%) died during the study treatment period, all secondary to hepatic failure; no other early deaths occurred. Two of these patients had baseline grade 1 bilirubin elevation (no other patients in the study reported baseline bilirubin elevations). Median follow-up was 21 months. The overall response rate was 50% (20/40) where 19 achieved CR while 1 achieved CRi. Of 20 responders, the median time to neutrophil count recovery was 28 days and to platelet recovery was 34 days. The median time to relapse was 22.6 months. The median overall survival (OS) of the entire cohort was 16 months (Figure 1). Conclusions: Len plus MEC was associated with a high response rate and improved OS in patients with R/R AML as compared to historical controls. The overall safety profile was similar to prior MEC chemotherapy trials but several patients did experience early hepatic toxicity. Overall these data support evaluating high-dose Len combined with MEC for patients with R/R AML in a randomized study.

Idarubicin and Cytarabine with and without Midostaurin for FLT3-Mutated Acute Myeloid Leukemia

Background: One of the standards of intensive induction therapy for young patients with acute myeloid leukemia (AML) consists of 7 days of cytarabine and 3 days of either idarubicin 12mg/m 2 (IDA) or daunorubicin 60-90mg/m 2 (DNR) (7+3). Midostaurin (MIDO) is added to 7+3 for patients with FLT3-mutated AML. The pivotal trial that led to the approval of MIDO (RATIFY) used 60mg/m 2 of DNR as the anthracycline of choice. Although a direct comparison of IDA with DNR 60mg/m 2 is lacking, randomized studies have shown comparable results with DNR 90mg/m 2 and IDA. It is unclear if the addition of MIDO to IDA-based induction leads to improved outcomes when compared to 7+3 (IDA) alone. Methods: We conducted a retrospective analysis to compare outcomes of young patients (≤65y) with FLT3-mutated AML treated with IDA-based induction, with and without MIDO, at the University of Alabama at Birmingham (UAB) between January 2010 and Dec 2022. Results: There were 85 patients included in the analysis (MIDO=46, control=39). The baseline characteristics are shown in Table 1. The median age of the MIDO and control groups was 51.5y (19-65y) and 50.5y (20-65y), respectively. In the MIDO group, more patients had FLT3-ITD (89% vs. 72%, p<0.01) and NPM1 mutation (52% vs. 33%, p<0.01) compared to the control group. There was an overrepresentation of intermediate karyotype in the MIDO group (92% vs. 76%, p<0.01) and favorable karyotype in the control group (4% vs. 15%, p<0.01). Majority of the patients received micafungin (75%) in the MIDO group and either voriconazole or posaconazole (77%) in the control group. All patients in the MIDO group received at least 1 dose and 70% of the patients completed all 14 days during induction. Ten patients had to discontinue MIDO early due to adverse events (gastrointestinal=6, hepatic=3, rash=1). The composite complete remission (cCR) rate was higher in the control group (79%) compared to the MIDO group (71%) (p=0.01). In comparison, the protocol specified CR rate (by day 60) in the RATIFY trial was 59%. There were no significant differences between the two groups in terms of time to neutrophil and platelet recovery, breakthrough invasive fungal infections, febrile neutropenia and length of hospital stay. In the MIDO group, 22 patients received MIDO during consolidation chemotherapy. Twenty-two out of 46 and 25/39 patients proceeded to allogeneic stem cell transplantation (allo-SCT) in the MIDO and control group, respectively. The 4-year overall survival (OS) for the MIDO group was 50%, compared to 51% for the control group (p=0.1) (Figure 1). The 4-year OS for patients <60y was 54% and 55% in the MIDO and control group, respectively. Conclusion: This is the first study to compare the impact of adding MIDO to IDA-based induction versus IDA-based induction alone. Our findings suggest that IDA-based induction may result in a higher cCR rate, with or without the addition of MIDO, when compared to DNR 60mg/m 2 (as per RATIFY). The addition of MIDO to IDA-based induction did not result in increased toxicity. These results suggest that IDA-based induction, instead of DNR 60mg/m 2, should be considered for younger patients with AML. The added benefit of MIDO, in the context of 7+3 with IDA or even DNR 90mg/m 2, needs further investigation.

Quantum-First: Safety By Treatment Phase and By Age in Newly Diagnosed (nd) Patients (pts) with FMS-like Tyrosine Kinase 3-Internal Tandem Duplication ( FLT3-ITD) Positive Acute Myeloid Leukemia (AML)

Background Results from the phase 3 QuANTUM-First study (NCT02668653) showed that in nd FLT3-ITD+ AML pts, the addition of the oral, highly potent, selective, type 2 FLT3 inhibitor quizartinib (Quiz) to standard chemotherapy ± allogeneic hematopoietic cell transplantation (allo-HCT), followed by Quiz monotherapy for up to 36 cycles (3 years [yrs]), decreased the relative risk of death by 22.4% vs placebo (PBO), with a generally manageable safety profile (PMID: 37116523). We describe safety by treatment phase (Induction [IND], Consolidation [CONS], Continuation [CONT]) and by age group (<60, 60-64, ≥65 yrs). Methods Eligible pts (18-75 yrs) with nd FLT3-ITD+ AML were randomized 1:1 to Quiz (40 mg/d) or PBO + standard IND chemotherapy. Pts in complete remission (CR) or CR with incomplete neutrophil or platelet recovery received standard CONS chemotherapy + Quiz (40 mg/d) or PBO and/or allo-HCT (Quiz/PBO not given with allo-HCT), followed by CONT of single-agent Quiz (30-60 mg/d) or PBO for up to 36 cycles. Safety was evaluated in pts treated with ≥1 dose of Quiz or PBO. Treatment-emergent adverse events (TEAEs) were coded by MedDRA v24.0, severity by NCI-CTCAE v4.03. Results In both arms, median treatment durations were 2 weeks in IND, 4 weeks in CONS, and 1.3 yrs in CONT. Rates of TEAEs and grade ≥3 TEAEs were similar in both arms in IND & CONS, although grade ≥3 TEAEs were more common with Quiz vs PBO in CONT (Table 1). In IND & CONS, the most common TEAEs were gastrointestinal (GI) AEs (diarrhea, nausea, vomiting), infections (pneumonia, sepsis), hypokalemia, pyrexia, febrile neutropenia, and rash. In IND & CONS, myelosuppression occurred in most pts in the Quiz and PBO arms, with a median blood count recovery time <4 weeks from the time of onset. In CONT, most common TEAEs were upper respiratory tract infections, GI AEs, cytopenias, and ALT increase. In CONT, more pts with Quiz had myelosuppression, with trends toward earlier onset and delayed recovery with Quiz. ECG QT prolonged was more common with Quiz vs PBO in all phases, QTcF >500 ms was low (2.3%) and observed in IND & CONS only. Ventricular arrythmias were few with Quiz (2 pts [0.8%] had cardiac arrest/ventricular fibrillation with severe hypokalemia). Infections were the most common serious TEAEs (TESAEs), with febrile neutropenia, pneumonia, and sepsis in IND & CONS and pneumonia and viral infections in CONT. TEAEs leading to death were numerically higher with Quiz vs PBO in IND & CONS, mainly due to infections in older pts, but numerically lower with Quiz vs PBO in CONT. In IND & CONS, TEAEs leading to dose interruptions or dose reductions were similar in both arms, whereas TEAEs leading to discontinuation (disc) were higher with Quiz vs PBO. TEAEs leading to dose interruptions, dose reductions, or disc in CONT were higher with Quiz vs PBO. TEAE leading to disc of Quiz were mostly infections in IND & CONS and cytopenias in CONT. Cytopenias and QT prolonged were the most common AEs leading to dose modifications and were more common with Quiz. There was no evidence of increasing toxicity with long-term Quiz therapy for up to 36 cycles in CONT, and no new AEs were observed in pts receiving >12 cycles. Rates of TEAEs and grade ≥3 TEAEs were similar in both arms across age groups (Table 2). TESAEs, TEAEs leading to death, and TEAEs leading to disc were more common in older pts (≥65 yrs [n=134]) vs younger pts (<60 and 60-64 yrs [n=399]) in both arms. Infections in the elderly were most commonly severe, serious, and fatal AEs and a main cause of more early deaths (<60 days of Quiz start). TEAEs leading to dose modifications were higher with Quiz vs PBO across age groups. In the Quiz arm, cytopenias, headache, rash, cough, ALT increase, hypomagnesemia, oropharyngeal pain, and back pain were more common in younger vs older pts; ECG QT prolonged was highest in the 60-64 yrs group. GI AEs and were more common in older pts with Quiz. QTcF >500 ms occurred mainly in pts 60-64 yrs with Quiz. Conclusions In QuANTUM-First, infections and cytopenias associated with Quiz were observed across phases. Fatal infections were more common with Quiz in IND & CONS but not in CONT. Rates of prolonged QTcF >500 ms were low overall, occurring mostly in IND & CONS, not in CONT. Pts ≥65 yrs had higher TESAEs, TEAEs leading to death (including early death), and TEAEs leading to disc (mainly infections) vs younger pts in both arms. Quiz safety profile in different treatment phases and age subgroups supports an overall positive benefit/risk.

The Impact of Baseline Genetic Profile and Treatment on Hematological Toxicity in CEBPA-Mutated Acute Myeloid Leukemia

Background: CEBPA-mutated acute myeloid leukemia (AML) is a separate entity in the updated WHO and ICC classifications. Previously identified by double mutations, the clinical profile and favorable outcome were correlated with in-frame mutations in bZIP domain. Beyond mutations recurrent in myeloid neoplasms (involving TET2, FLT3, NRAS), CEBPA-mutated AML is enriched in gene mutations (i.e., GATA2, CSF3R, WT1) rarely observed in CEBPA-wild type AML. No conclusive findings have been drawn for the prognostic impact of additional mutations. Of note, somatic GATA2 mutations have been associated with invasive fungal infections in myeloid neoplasms. Although overall correlating with favorable prognosis, an unexpectedly high rate of treatment-related mortality (TRM) in CR has been described in a large multicenter study on intensively treated CEBPA-mutated patients (pts) (Schlenk et al Blood 2013;122(9):1576). Aims: The aim of our study was to correlate baseline and treatment characteristics with the outcome and hematological toxicity in an intensively treated cohort of CEBPA-bZIP mutated AML pts. Methods: Pts entering the study had a diagnosis of CEBPA-bZIP AML confirmed according to 2022 WHO and ICC criteria. Pts were characterized by next-Generation deep amplicon sequencing with Ion Torrent platform (ThermoFisher Scientific) covering a panel of 40 genes. Sequence alignment and filtering were performed by NextGENe version 2.4.2.1 (SoftGenetics). The first two chemotherapy cycles were categorized according to the delivery of an anthracycline (ANTHRAC) and cytarabine (ARA-C) at high dose (HDAC). The study was approved by the local institutional review board. Results: From 2004 to 2023, 49 pts with CEBPA-bZIP mutated AML met the inclusion criteria at project study sites (Firenze, Bergamo). Their features are detailed in Table 1. CR rate was achieved in 45 of 49 pts (91.8%), with a DFS and OS of 74.3% and 82.4% at 5 y, respectively. Based on NGS, at least one additional mutation was identified in 18 (36.7%) pts (NGS+), the majority of them involving GATA2 (n=14, 28.6%). As per treatment, 44 pts received at least two cycles, of which no (n=2, 4.6%), one (n=14, 31.8%), or two (n=28, 63.6%) ANTHRAC-containing cycles. As regarded HDAC, 19 (43.2%), 17 (38.6%) and 8 (18.2%) pts received no, one or two HDAC-containing cycles, respectively. No significant differences emerged for DFS or OS between NGS+ and NGS- groups (P=0.33 and 0.52, respectively). To gain insight into potential causes of toxicity during CR, we focused on hematopoietic recovery after induction and consolidation. NGS+ pts showed slower neutrophil (ANC) recovery (median, 22 days to >500/uL after induction and 25 days after consolidation) compared to NGS- group (19 days, P=0.018; 18 days P=0.058, respectively). Such effect was enhanced by treatment with ANTHRAC-containing cycles: NGS+ pts receiving two cycles required significantly longer time to recover from first consolidation cycle, both for ANC (30 days) and platelet count (34 days) vs 22 (P=0.012) and 27 (P=.010) days, respectively, of other categories (Figure 1). A proportion of 45.5% (5/11) NGS+/ANTHRAC-2 pts could not complete the planned chemotherapy program due to hematological toxicity compared to 19.3% (6/31, P=0.09) in other categories. No significant prolongation of recovery was observed for HDAC. Conclusions: CEBPA-bZIP mutated AML is featured by high response rate and favorable outcome. Delayed hematopoietic recovery was observed in pts bearing additional mutations (especially GATA2) when treated with anthracyclines in first two cycles. Our findings suggest the cumulative dosage of anthracyclines should be limited in CEBPA-mutated/NGS+ patients to spare hematological toxicity on turn impairing the therapeutic plan.

Inotuzumab Ozogamicin + Blinatumumab +/- Rituximab + Mini-Hcvd Shows Improved Tolerability with Similar Efficacy Compared to E1910 in Treatment of Philadelphia Chromosome Negative Acute Lymphoblastic Leukemia in Older Adults

CONCLUSION Philadelphia chromosome negative acute lymphoblastic leukemia (Ph- ALL) boasts contradicting survival rates given its binodal age distribution. While children and adolescents post 5-year overall survival (OS) rates of 89%, the stark contrast of 5-year OS of 20.4% in older adults greater than 65 years, makes this a challenging population to treat. Currently, there is no standard of care for the treatment of ALL with various regimens utilized dependent on age, co-morbidities, performance status, as well as physician/institute preference. One regimen that is commonly used is a combination of targeted therapies along with reduced intensity chemotherapy (inotuzumab ozogamicin, rituximab (if CD20+), blinatumomab and mini hyper fractionated cyclophosphamide, vincristine, and dexamethasone [Mini-HCVD]), which has demonstrated both improvements in survival while reducing treatment related toxicity. Another commonly accepted approach is a modified Berlin-Frankfurt-Munster (BFM) backbone (i.e., E1910). Despite the available options, there is a lack of comparison of these regimens. To answer this unanswered question, we compared the characteristics, outcomes, and toxicities of older adults with ALL treated with either Mini-HCVD or E1910 at our academic medical center. Medical records were retrospectively reviewed at Roswell Park Comprehensive Cancer Center to identify patients diagnosed with Ph- ALL treated with Mini-HCVD or E1910 between January 1 2016 - May 31 2022. Those with relapsed/refractory ALL or received E1910 in the setting of a clinical trial were excluded from the study. Demographics, disease-specific variables, and overall outcomes were collected in accordance with the institutional review board approved protocol. Primary outcome was 3-year overall survival rate, while secondary outcomes included best response in first 3 months, minimal residual disease (MRD) status at 3 months, 30-day mortality, as well as the frequency and incidence of toxicities per cycle. Best response was defined as less than 5% blasts on bone marrow biopsy with complete (CR) or incomplete (CRi) absolute neutrophil or platelet count recovery. MRD status was assessed via flow cytometry and next generation sequencing using ClonoSEQ®. Adverse events and their severity were characterized according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. Differences in OS between two groups were examined by log-rank test. Propensity scores were obtained based on the baseline variables that with potentially imbalanced distributions (p<0.1). MRD status and response to treatment were compared using Fisher's exact test. Toxicities were summarized as frequencies for each treatment cycle. The incidence of grade 3-4 toxicities was defined as the number occurrence per treatment cycle and were compared using Poisson regression. 27 patients were included, with 15 (55.5%) receiving Mini-HCVD and 12 (44.5%) receiving E1910. Median age was 70 years (49-83), in Mini-HCVD arm and 63 years (47-74), in E1910 arm. Three-year OS was 61.9% for those who received Mini-HCVD (95% CI 40.2-95.3) and 28.1% for those who received E1910 (95% CI 10.9-72.9; p=0.23). In Mini-HCVD 77% and 69% of patients achieved CR/CRi and MRD negative status in the first three months, compared to 60% and 70% in E1910. Only 13% of patients in Mini-HCVD proceeded to transplant or CAR-T, compared to 58% in E1910 (p=0.0198). The most common treatment-related adverse events in both arms were cytopenias. Febrile neutropenia occurred in 47% of Mini-HCVD patients compared to 83% in E1910. There was no difference in rates of infections between arms. Mean incidence of grade 3/4 toxicity per cycle was 0.85/0.96 with Mini-HCVD and 1.03/1.65 with E1910 (p=0.0098). This retrospective single institute study demonstrates that the clinical outcomes of Mini-HCVD plus immunotherapeutic agents in older adults with Ph- ALL are similar to modified BFM backbone with reduced incidence of toxicity. While limited by small numbers, these results suggest that reduced intensity chemotherapy in combination with novel targeted agents could represent a new standard of care regimen in this historically difficult to treat patient population.

Cladribine and Cyclophosphamide Lymphodepletion Prior to Brexucabtagene Autoleucel in Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia and Mantle Cell Lymphoma

Introduction : Fludarabine and cyclophosphamide (Flu/Cy) lymphodepletion (LD) prior to chimeric antigen T-cell (CAR T) infusion favorably impacts CAR T expansion and efficacy. A national shortage of Flu prompted our center to explore substitute Flu with Cladribine (Cla) as part of Cla/Cy LD. Here, we report our experience with this regimen prior to brexucabtagene autoleucel (brexu-cel) for patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) and mantle cell lymphoma (MCL). Methods : We performed a retrospective, single-center analysis of consecutive patients who received brexu-cel for RR B-ALL and MCL at Moffitt Cancer Center (1/2022 and 4/2023). The Cla/Cy LD regimen utilized Cy at 500 mg/m 2 x 3 days for MCL or 900 mg/m 2 x 1 day for B-ALL (two patients received MCL dosing) and substituted Flu 30 mg/m 2 with Clad 5 mg/m 2 x 3 days. We compared best overall response (BOR), including measurable residual disease (MRD) status by the clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) ± PCR for B-ALL, event-free survival (EFS, event = progression or death) and overall survival (OS) in those receiving standard Flu/Cy vs Clad/Cy LD. CRS and ICANS were graded as per ASTCT criteria. For patients with a sustained response ≥6 months and who did not undergo stem cell transplant, we assessed serial absolute neutrophil counts (ANC), CD4, CD8, CD19 counts and IgG levels post-CAR T. The HEMATOTOX score (HS, Rejeski et al, 2021) was used to stratify patients for risk of post-CAR T cytopenias. Results : We analyzed 63 patients who received brexu-cel: 23 with ALL (10 Cla/Cy) and 40 with MCL (7 Cla/Cy). Rates of CRS grade ≥ 3 (12% vs Flu/Cy 4%; p=0.29) and ICANS grade ≥ 3 (24% vs Flu/Cy 40%; p = 0.25) did not differ with LD. Two ALL (1 Flu/Cy, 1 Clad/Cy) and one MCL (Flu/Cy) patients died prior to day 28 reassessment. BOR did not differ by LD and histology (Figure 1). MRD negativity in ALL was also comparable (Cla/Cy 5/7, Flu/Cy 10/11, p=0.52). Among ALL patients, 5 received alloSCT consolidation (1 Cla/Cy). Progression was noted in 3/8 Cla/Cy and 6/11 Flu/Cy patients (p=1.0). Fifteen ALL patients experienced early infections (6/10 Cla/Cy, 9/13 FluCy, p=0.69), including bacterial (5), viral (4), and fungal (3). Three deaths (2 Cla/Cy) occurred before day 100, all due to refractory fungemia post anakinra. Two later deaths were attributed to relapse (both Flu/Cy). Among MCL patients, progression was noted in 16 (1 Cla/Cy). Seven MCL patients experienced early infection (all Flu/Cy), including bacterial (6), fungal (2), and viral (1). Two deaths occurred before day 100, 1 related to CRS and fungemia post anakinra (Flu/Cy), and another from stroke (Cla/Cy). Four (all Flu/Cy) have developed a second cancer (2 MDS/AML, 1 prostate, 1 liver), and 1 each died in remission from late infection (sepsis) and intracranial hemorrhage (both Flu/Cy). Cla/Cy patients had higher median ANCs at 28, 90, and 180 days (Table 1). When only patients with HS ≥ 2 were considered, 28-day ANC remained higher after Cla/Cy LD (1.57 vs Flu/Cy 0.67; p=0.04), numerically higher at 90 days (2.36 vs Flu/Cy 1.82; p=0.38) and again significantly higher at 180 days (3.96 vs Flu/Cy 1.2; p=0.05). Median CD4 was higher for Cla/Cy patients at day 28 (0.232 vs Flu/Cy 0.121; p=0.04), and numerically higher at day 90 (0.244 vs Flu/Cy 0.152; p=0.31) and 180 (0.254 vs Flu/Cy 0.161; p=0.21). Median CD8 was similar at day 28 (0.197 vs 0.155; p=0.46), 90 (0.435 vs Flu/Cy 0.167; p=0.65), and 180 (0.557 vs 0.220; p=0.14). There was no difference in IgG levels at day 28 (502 vs Flu/Cy 466; p=0.87), 90 (385 vs Flu/Cy 393; p=0.76), or 180 (424 vs Flu/Cy 426; p=0.94). Median CD19 was greater for Cla/Cy at day 90 (median 0.095 vs 0.000; p<0.01) and numerically higher at day 180 (0.213 vs Flu/Cy 0.000; p=0.29). Median duration of followup was 245d for ALL (FluCy 361d, ClaCy 194d) and 746d for MCL (FluCy 812d, ClaCy 215d). Median DOR, EFS, and OS did not differ by LD (Figure 1). Conclusions : Cla/Cy LD prior to brexu-cel is feasible in ALL and MCL patients with comparable efficacy. Toxicity rates including CRS, ICANS and early infections were similar to those seen with Flu/Cy, but there was a trend towards earlier recovery of neutrophils, B-, and T-cells with Cla/Cy. The trend was demonstrable even in patients at high risk of prolonged post-CAR T neutropenia as identified by the HS. Longer follow-up and correlative analyses are warranted to further explore differences between Cla/Cy and Flu/Cy.

15-Pgdh Inhibition Alternatively Activates Macrophages to Promote Hematopoietic Function during Aging

Aging results in the progressive decline of organ function and integrity across species. In the hematopoietic system aging is associated with diminished stem cell regenerative capacity and contributes to compromised tissue homeostasis and heightened vulnerability to age-related diseases including anemia, myelodysplastic syndromes, and hematological malignancies. Macrophages, integral components of the innate immune system, demonstrate remarkable functional plasticity and play diverse roles in tissue homeostasis, inflammation, and immunity. The balance between pro-inflammatory M1 and anti-inflammatory M2 macrophages significantly influences immune and inflammatory responses, and thus the progression of hematopoietic aging. We have previously demonstrated that the prostaglandin degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) regulates hematopoietic stem cell (HSC) activity, that 15-PGDH inhibition (PGDHi) following transplantation results in a markedly accelerated recovery of neutrophils, platelets, and HSCs, and that 15-PGDH knockout animals display enhanced HSC function with age. Here we expand on our previous work and demonstrate in aged mice that (1) 15-PGDH expression and activity remains conserved (2) that PGDHi induces a gene expression signature consistent with alternatively activated M2 macrophages, (3) that prolonged PGDHi results in a significant increase in phenotypic HSCs in both the bone marrow and spleen and induces a gene signature consistent with enhanced HSC function, and (4) that PGDHi treated bone marrow (BM) displays a advantage during primary competitive repopulation studies and prevents age-associated myeloid bias during secondary transplants. Taken together we propose PGDHi represents an in vivo switch to alternatively activate macrophages, which has significant implications to not only prevent age-associated hematopoietic decline, but also as a novel strategy to treat age-associated hematologic disease.

Mitoxantrone Hydrochloride Liposome Injection Combined with Carmustine, Etoposide, and Cytarabine As Conditioning of Autologous Hematopoietic Stem Cell Transplantation in NHL Patients：a Prospective Single-Arm Clinical Trial

Background：High-dose chemotherapy and autologous stem-cell transplantation (ASCT) is standard consolidation therapy for aggressive lymphoma.Choosing the most effective conditioning regimen prior to ASCT can lead to longer survival in these patients.Mitoxantrone liposome is an anthraquinone antibiotic anti-tumor drug. Previous studies have shown that mitoxantrone liposome monotherapy or combined with other drugs has achieved good efficacy for T-cell lymphoma, but there are no reports of its application in conditioning. Objective:This is a prospective single-arm clinical study to evaluate the efficacy and safety of mitoxantrone hydrochloride liposome injection combined with carmustine, etoposide, cytarabine (modified BEAM protocol) as conditioning of autologous hematopoietic stem cell transplantation for NHL patients (NCT05681403). Methods: Patients with non-Hodgkin lymphoma who planned to undergo ASCT were eligible to participate in the trial if they were 18 to 60 years of age.The patients received mitoxantrone hydrochloride liposome injection 24 mg/m 2,ivgtt,d-7, carmustine 300 mg/m 2,ivgtt,d-6, etoposide200 mg/m 2,ivgtt,d-5~ -2,cytarabine 200 mg/m 2 q12h,ivgtt,d-5~ -2 as conditioning regimen.The primary end point was the relapse rate within three years and the secondary end point was adverse events of modified BEAM protocol. Results：Twenty-five NHL patients (median age 39 years old,17 males and 8 females) were enrolled,including 40% DLBCL, 12% MCL, 4% primary mediastinal B-cell lymphoma,4%B lymphoblastic lymphoma,4%Marginal zone B-cell lymphoma,12% PTCL-NOS, 8% ALK +ALCL,8% NK/T lymphoma, 8% AITL,4% ALK -ALCL and 4% Sézary syndrome. 18 cases achieved complete remission before ASCT, while 7 cases received partial remission before ASCT. The median of neutrophil recovery time was 11 days(7-15) and the median of platelet recovery was 12days (7-60).Nausea,vomit,constipation,blue skin and cough were the most frequently reported conditioning related AEs (88%,16%,12%,12% and 8% of patients,respectively), and other AE included 1 case of hypoxemia, 1 case of hypotension, 1 case of palpitation, 1 case of rash. Except one patient experienced a grade 3 vomit ,the others were grade 1-2 and improved after symptomatic treatment. Nine patients experienced febrile neutropenia and 2 cases of sepsis, 4 cases of respiratory infection, and 3 cases of oral mucositis. All partial remission patients achieved complete remission post-HSCT and one of them relapsed 7 months after ASCT.The others were all in complete remission during follow-up. Conclusion:Mitoxantrone hydrochloride liposome injection combined with carmustine, etoposide, cytarabine (modified BEAM protocol) was a well-tolerated and effective conditioning regimen for NHL and the study is ongoing and updated results will be presented in the future.

Local Oxygen Dictates Hematopoietic Cell Growth and Function

Functional hematopoietic stem (HSC) and progenitor cells (HPC) are responsible for the replenishment of blood/immune cells, are a source of treatment for diseases, and when dysregulated are a source of hematological neoplasia. To identify factors that impact HSC/HPC functional competency, we performed a transcriptomic screen which revealed that human cord blood (CB) HSCs/HPCs capable of early engraftment in an NSG mouse model of transplantation exhibited enriched expression of gene programs that vary with changes in oxygen (O 2) availability compared to non-transplanted input cells. This is physiologically and clinically relevant as HSCs/HPCs and diseased hematopoietic cells such as acute myeloid leukemia (AML) are exposed to O 2 tensions ranging from <1% to 21% as they function in various anatomical sites or are harvested for clinical applications. Thus, there are translational and practical rationales for understanding the growth and functional properties that hematopoietic cells exhibit across the range of O 2 they experience physiologically or artificially, which we examined here. First we found significantly different frequencies of HSCs/HPCs and colony forming units (CFU) in human bone marrow (BM), mobilized peripheral blood (PB), and CB, which have varied O 2 tensions, suggesting O 2 availability may impact hematopoiesis. To directly examine this, human CB CD34+ cells were split and plated in CFU assays or expanded in growth stimulating conditions at 1% O 2, 3% O 2, 5% O 2, 14% O 2 (physiologic tensions), or 21% O 2 (processing tension) . HPCs expanded best and formed more differentiated CFUs in high physiologic O 2 tensions and extra physiologic O 2 tensions, while HSCs expanded better and more potent CFUs were formed in a mid-physiologic O 2 tension (5%). This suggests that O 2 dependent pathways are critical to HSC/HPC growth but there may be a “sweet spot” for early HSCs/HPCs that allows for proliferation while maintaining self-renewal and potency to maintain a pool of primitive cells, possibly mirroring in vivo O 2 responses. In in vivo functional HSC/HPC analyses, NSG mice transplanted with CB units expanded in physiologic O 2 exhibited significantly better early neutrophil recovery and long-term myeloid reconstitution compared to input, suggesting that growth and maintenance of HSC/ myeloid HPC functional competency is better balanced in physiologic O 2 compared to extra physiologic tensions. Platelet recovery and lymphoid reconstitution were significantly better in mice receiving uncultured control CB, suggesting expansion does not affect all HSC/HPC subpopulations equally. Mechanistically, cells grown in low O 2 tensions (1-3%) had higher frequencies of early HSCs/HPCs, significantly lower intracellular ROS levels, a significantly higher percentage of cells in cell cycle G0, and lower levels of phosphorylated γH2AX than those grown in higher O 2. These data suggest that while to some extent HSCs/HPCs require O 2 to proliferate, they also exhibit properties associated with exhaustion when maintained in higher O 2. We next examined the effects varying O 2 tensions have on diseased hematopoietic cells using primary mouse AML cells or oncogene transformed human CB CD34+ cell lines. AML cells proliferated significantly more rapidly and formed more CFUs in higher O 2 tensions, suggesting that O 2 sensing pathways may be a dependency in AML that could affect disease progression as local O 2 tensions become perturbed. AML cells grown in lower O 2 exhibited lower resting levels of phosphorylated γH2AX (measured by flow cytometry) and lower O 2 consumption rates (measured by Seahorse metabolic flux analyzer), suggesting they are less susceptible to cellular stress. In line with this, AML cells maintained in low O 2 exhibited significantly better proliferation and recovery after treatment with the chemotherapeutic cytarabine compared to those in higher O 2 tensions. Thus, diseased cells growing in lower O 2 niches may be less susceptible to treatment and therefore may be a source of chemoresistance or relapse. These data reveal important roles for O 2 in balancing hematopoietic cell growth with normal and diseased cell function. Manipulating O 2 tension or O 2 sensing pathways could be used to improve HSC/HPC expansion for transplantation or to treat disease. Transcriptomic studies of HSCs/HPCs and AML cells are ongoing to determine the O 2 sensing mechanisms that dictate cellular function.

Post-Transplant Cyclophosphamide-Based Graft-Versus-Host Disease Prophylaxis in HLA-Matched and Haploidentical Donor Transplants for Patients with Hodgkin disease: A Comparative Study of the LWP EBMT

INTRODUCTION Post-transplant cyclophosphamide (PTCy) has proven to be a highly effective strategy in preventing graft-versus-host disease (GVHD) in haploidentical (haplo) hematopoietic stem cell transplantation (HSCT), but it is being increasingly used in HLA-matched transplants. The outcomes of haplo HSCT with PTCY in various hematologic malignancies, particularly in relapsed/refractory Hodgkin disease (HD), have shown promising results, challenging those obtained in transplant patients from HLA-matched donors without PTCy. However, there is limited information on the impact of donor types on the outcomes of patients with HD undergoing allogenic HSCT when using homogeneous GVHD prophylaxis with PTCy. To address this knowledge gap, we have conducted an extensive study using the Lymphoma Working Party of the European Society for Blood and Marrow Transplantation (LWP-EBMT) registry. The aim of this study was to compare the outcomes of patients with HD undergoing HSCT from both HLA-matched donors, which include matched sibling donors (MSD) and matched unrelated donors (MUD), and haplo donors, using PTCy as GVHD prophylaxis approach in all cohorts. PATIENTS AND METHODS Inclusion criteria All adults (≥ 18 years) patients diagnosed with HD who underwent their first HSCT between January 2010 and December 2020 from either a MSD, MUD or haplo, and whose data were reported to the EBMT registry were included. For the purposes of this study, only transplants who received PTCy-based as GVHD prophylaxis were considered. Statistical Analysis The study utilized statistical methods such as Kaplan-Meier estimation, Log-Rank test, competing risks analysis, and Cox proportional hazards regression. The analyses included OS, PFS, NRM, aGVHD, cGVHD, and graft failure. The statistical software R and packages were used. RESULTS Patient and Transplantation Characteristics Patient, disease and transplant-related characteristics of the 860 patients are summarized in Table 1. Engraftment The cumulative incidence (Cum Inc) of neutrophil recovery at day 30 was 95% (95% CI, 93-96) in the HLA-matched group and 96% (95% CI, 91-98) in the haplo cohort (p=0.18). The 60-day Cum Inc of platelet recovery in similar order was 94% (95% CI, 88-97) and 86% (95% CI, 83-88) (p<0.001). GVHD The Cum Inc of acute GVHD grade II-IV at 100 days in the HLA-matched and the haplo cohorts was 24% (95% CI, 17-31) and 34% (95% CI, 30-37), respectively (p=0.01), whereas for grade III-IV it was 8% (95% CI, 4-13) and 10% (95% CI, 8-13), (p=0.44). In multivariable analysis (table 2), haplo was associated with an increased risk of acute GVHD grades II-IV, when compared with HLA-matched (HR 0.65; 95% CI, 0.46-0.93; p=0.01). Other variables are shown in table 2. The 2-year Cum Inc of chronic GVHD in the HLA-matched and haplo cohorts was 26% (95% CI, 19-33) and 27% (95% CI, 24-31), respectively (p=0.75). Other variables are shown in table 2. NRM and relapse The Cum Inc of NRM at 2 years was 10% (95% CI; 6-16) for HLA-matched and 18% (95% CI; 15-21) for haplo (p=0.02). In multivariable analysis (table 2), haplo was associated with an increased risk of NRM, when compared to HLA-matched (HR 0.5; 95% CI, 0.28-0.89; p=0.01). Other variables are shown in table 2. The Cum Inc of relapse at 2 years was 22% (95% CI; 15-29) for HLA-matched and 24% (95% CI; 20-27) for haplo (p=0.81). Other variables are shown in table 2. Survival The 2-year OS for the HLA-matched and haplo cohorts was 82% (95% CI; 75-88) and 70% (95% CI; 67-74), respectively (p=0.002). In multivariable analysis (table 2), compared to HLA-matched, haplo (HR, 0.51; 95% CI, 0.34-0.77; p=0.001) was associated with worse survival. Other variables are shown in table 2. The 2-year PFS was similar between HLA-matched and haplo (66%; 95% CI, 58-74; and 58%; 95% CI, 54-62, respectively; p=0.17). Other variables are shown in table 2. Comparison of outcomes for MSD and MUD With regards to the donor type, most posttransplant outcomes did not exhibit significant differences, except for a higher incidence of grades II-IV acute GVHD in the MUD group (33%; 95% CI, 32-44) compared to the MSD group (17%; 95% CI, 10-25) (p=0.01). CONCLUSION Our study shows that in patients with HD undergoing HSCT with PTCy for GVHD prophylaxis, no significant differences in 2-year PFS were observed between HLA-matched and haplo. A higher risk of acute GVHD and NRM is associated with haplo, leading to lower OS compared to HLA-matched transplantation.

Comparison of Safety and Efficacy between Venetoclax Combined with CAG (cytarabine, aclarubicin, G-CSF) Regimen and CAG Regimen Alone in Patients with Relapsed or Refractory Acute Myeloid Leukemia

Background: Patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) have dismal outcomes, representing an urgent unmet need. The BCL2 inhibitor venetoclax has exhibit encouraging anti-tumor activity in AML. The clinical efficacy of the CAG regimen in patients with R/R AML has been confirmed in previous studies. The aim of this study was to retrospectively compare the safety and efficacy of the combination of venetoclax with CAG regimen and CAG alone in patients with R/R AML. Patients and methods: We retrospectively reviewed and compared the efficacy and safety of venetoclax combined with the CAG regimen (Ven-CAG group, n=37) and CAG regimen alone (CAG group, n=37) for 74 patients with R/R AML treated at our institution between August, 2016 and March 2023. The patients included those who were refractory to at least one cycle of induction chemotherapy, and patients with relapsed leukemia. According to the duration from complete remission (CR) to relapse, the relapsed patients were further divided into two subgroups, the favorable-risk relapsed group (the duration≥12 months) and the poor-risk relapsed group (the duration<12 months). The incidence of composite complete remission (CCR), minimal residual disease (MRD) status, one-year overall survival (OS), and adverse events (AEs) were analyzed and compared in two groups. Results: Patient age, sex, ELN risk stratification, disease status, risk stratification according to the duration from CR to relapse were comparable between two groups (Table 1). The CCR of Ven-CAG group was 78.4% (29/37)，which was superior to 56.8% (21/37) of CAG group (P=0.047) . Furthermore, the rate of MRD elimination in patients who achieved CCR in Ven-CAG group was significantly higher than that in CAG group [44.8% (13/29) vs. 9.5% (2/21), P= 0.007] (Table 2). The rate of MRD elimination in favorable-risk relapsed group (CR to hematological relapse ≥12 months) was higher in Ven-CAG group [100% (5/5) vs. 25% (1/4), P= 0.048]. The one-year OS were 78.4% (95% CI, 62.3%-94.5%) and 66.7% (95% CI, 51.2%-82.2%) in Ven-CAG and CAG group (P=0.225), respectively. The incidences of grade 3-4 hematologic AEs in Ven-CAG group and CAG group were 100% (37/37) and 97.3% (36/37), respectively (P=1.000). The incidences of febrile neutropenia were 67.6% (25/37) and 62.2% (23/37), respectively (P=0.626). Among patients who achieved CCR, the median time for neutrophil recovery in the Ven-CAG group and CAG group were 8 (3-30) days and 9.5 (3-31) days (P=0.781), while the median time for platelet recovery was 9 (3-28) days and 8 (3-19) days (P=0.708). The incidences of grade 3-4 non-hematologic AEs were comparable in Ven-CAG and CAG group (32.4% vs. 35.1%, P=0.806). The most common nonhematologic AEs were infections, with the incidences of 18.9% and 27%, respectively (P=0.407). No patients died within 4 weeks after initiating the induction course in Ven-CAG group. There was one case of early death during induction in CAG group due to cerebral hemorrhage. Conclusions: Venetoclax added to CAG regimen was safe and active in patients with R/R AML, producing higher CCR and MRD-negative remissions than CAG regimen alone. These results highlight the incremental benefit of venetoclax added to CAG regimen. This requires further verification through prospective, larger size clinical trials.

Fractionated Total Body Irradiation and Fludarabine Based Conditioning Regimen with Post-Transplant Cyclophosphamide (PTCy) for Mismatched Related and Unrelated Donors HCT for Acute Leukemia and MDS

Background: Allogeneic hematopoietic cell transplantation (alloHCT) remains the only curable treatment option for several hematological malignancies. Preparative and graft-versus-host disease (GVHD) prophylactic regimens combined with graft-versus-leukemia effect (GVL) are important in preventing graft rejection, disease relapse and GVHD; and subsequently impact the success of this life saving procedure. Choice of regimen depends on patient, donor/graft source and disease characteristics. Objectives: Assess the outcomes of patients who underwent haploidentical (haplo) or mismatched unrelated donor (MMUD) HCT with myeloablative conditioning (MAC) regimen using radiation in combination with fludarabine (FLU) with PTCy as higher intensity GVHD prophylaxis and evaluate the feasibility of a fludarabine and fractionated total body irradiation (FLU/FTBI)-based regimen in both donors. Study Design: Retrospective analysis to study the outcomes of patients receiving FLU/FTBI as myeloablative conditioning for mismatched (related or unrelated) donor HCT combined with post-transplant cyclophosphamide (PTCy) as GVHD prophylaxis. Results: One hundred and fifty-five consecutive patients undergoing HCT at City of Hope (COH) from January 2015 to December 2021 were included. Median age was 38 years (range 9-60) and 56.8% of patients were male and 97.5% had a Karnofsky performance status (KPS) ≥ 80. HCT comorbidity index was ≥ 3 in 36.1% cases and 29% with a disease risk index (DRI) of high/very high. Disease diagnosis included acute lymphoblastic leukemia (ALL) (46.5% of cases) and acute myeloid leukemia (AML) (36.1% of cases). The median donor age was 31-years with 67.1% haplo donors and 32.9% mismatched unrelated donors (MMUD). MMUD HCT led to faster neutrophil recovery (15 vs 16 days, p= 0.01) and platelet count recovery (18 vs 23 days, p= 0.029). Day 100 cumulative incidence of grade II-IV and III-IV acute GVHD had no statistical difference between the two cohorts. With a median follow-up of 24 months (range 3 to 81), 100-day and 1-year NRM was 1.9% and 8.5%, respectively. There was no difference in the NRM between MMUD and haplo (HR 0.92, CI (0.35,2.39), p=0.86). Two-year relapse rate and DFS were 11.8% and 76.9%, respectively. In this study, 7 out of 51 patients in the MMUD group and 17 out of 104 patients in the haplo group relapsed with no statistically significant difference between the groups (HR 0.82, CI: (0.35,1.97), p=0.67). At a median follow-up of 2 years, GRFS was 60% (95% CI 0.51- 0.67). There was no statistically significant difference in GRFS between the MMUD and haplo groups (HR 0.76, CI (0.44,1.32), p=0.33). Two-year OS was 80.1%. There were no DFS or OS differences between the MMUD and haplo groups. On multivariate analysis, age less than 40 and low to intermediate DRI showed a DFS benefit (p = 0.004 and 0.029 respectively). None of the demographic factors affected OS or GRFS. Disease risk index was the only factor on multivariate analysis affecting relapse rates with higher risk of relapse with higher DRI (p= 0.017) and extensive chronic GVHD risk (p= 0.014) with no impact on acute GVHD. Conclusions: FLU/FTBI followed by PTCy for GVHD prophylaxis is a well-tolerated myeloablative regimen in mismatched donors with acute leukemia or myelodysplastic syndrome (MDS) with low transplant related morbidity and mortality and has produced promising HCT outcomes.

Update Results of a Phase II Trial of Venetoclax in Combination with Azacitidine and Chidamide in Relapsed/Refractory Acute Myeloid Leukemia

Background: Therapies for relapsed or refractory (R/R) AML remain limited, with low response rates and short duration of response, especially in patients ineligible for intensive cytotoxic chemotherapy. Combinations of venetoclax (VEN), chidamide (CHI) plus azacytidine (AZA) have shown to be synergistic with low intensity and intensive chemotherapy in preclinical data and in relapsed or refractory (R/R) setting (Phase II, 2022 ASH). Here we present updated results of this Phase II trial. Methods: Eligible patients were >18 yrs old with R/R AML, defined as lack of response rate after at least one cycle of induction or relapsed disease with any prior number of treatments. Patients received AZA 100mg on D1-7, VEN 200mg on D1-14, and CHI 30mg biweekly in 28-day cycles. The primary objective was evaluation of CR/CRi. Secondary objectives included event-free survival (EFS), overall survival (OS) and adverse events (AEs). Clinical responses were determined using the 2017 ELN criteria. Results: Between January 2022 and April 2023, 53 patients with R/R AML were enrolled in the study. The median age was 51 years (range 18-73). This regimen was salvage 1 in 79% (42) of patients, salvage 2 in 11% (6) and salvage 3 in 9% (5). 17%, 36%, and 47% of patients were classified as favorable, intermediate, and adverse ELN risk, respectively ( Table 1). 11% of patients had TP53 mutation, 22% had DNMT3A mutation, 21% had FLT3-ITD mutation, 9% had ASXL1 mutation, 19% had NPM1 mutation, 9% had IDH1 mutation, 11% had IDH2 mutation ( Fig. A). Among the 53 patients, the ORR of these patients was 68% with CRc rate of 53% after one cycle, and 81% with CRc rate of 72% after two cycles ( Fig. B). By ELN risk, ORR and CRc rates were 70% and 50% for favorable risk, 40% and 40% for intermediate risk, and 43% and 30% for adverse risk. Pretreatment IDH1 and IDH2 mutations were associated with higher CRc rates (75% and 50%, respectively). The CRc rate in patients with TP53 mutations was 67%, with response noted among patients with concurrent IDH1/ IDH2 mutations. With a median follow-up of 6.7 months, the median EFS and OS were 6 (95%CI 3.3-8.6) months and not reached (NR), respectively. Among patients with CRc, the 6-month EFS and OS were 64% and 88%, respectively, among those receiving this regimen as first salvage had encouraging outcomes (median EFS, NR) ( Fig. C-D). Of the 8 patients that proceeding with allogeneic hematopoietic stem cell transplant (allo-HSCT), the median EFS and OS were not reached after transplant ( Fig. E-F) No non-hematologic AEs of grade ≥3 were observed. The most common grade 1-2 non-hematologic AEs included nausea (28%), fatigue (28%), hypokalemia (17%), hypoproteinemia (17%), elevated transaminase levels (33%) and hyperbilirubinemia (11%). The occurrence rates of grade 3/4 hematologic adverse events (AEs) were as follows: white blood cell decrease (87%), median time to white blood cell recovery 23 (range 4-29) days; neutropenia (96%), neutropenia with fever (17%), median time to neutrophil recovery 25 (range 10-29) days; platelet decrease (68%), median time to platelet recovery 28.5 (range 7-44) days; anemia (64%), median time to hemoglobin recovery 29 (range 13-56) days. 30-day and 60-day mortality were 0% and 5.7%, respectively. 3 deaths occurred within 60 days in patients with persistent leukemia. Conclusions: Thecombination of VEN and CHI plus AZA (VCA) demonstrated manageable safety and encouraging efficacy in patients with R/R AML. IDH1/ IDH2 mutations were associated with the sensitivity of VCA, even in some patients with concurrent TP53 mutations.

Outcomes and Late Complications after Umbilical Cord Blood Transplantation for Fanconi Anemia: A Study on Behalf of Eurocord and Severe Aplastic Anemia Working Party - EBMT

Fanconi Anemia (FA) remains a disease with poor prognosis for which hematopoïetic cell transplantation (HCT) offers the only available curative option, with particular good results observed after matched sibling donor transplants. This study aimed to assess transplant outcomes and describe late complications in a large cohort of patients with FA who underwent umbilical cord blood transplantation (UCBT) in the European Society for Blood and Marrow Transplantation (EBMT) affiliated centres. Methods: We retrospectively reviewed all cases of FA whoreceived UCBT as first allogeneic transplant between 1988 and 2021. Data on late effects were collected with a questionnaire completed by transplant centres for patients who survived >2 years (y) after UCBT. Results: A total of 205 patients with FA received UCBT (55 related and 150 unrelated) in 77 transplant centres. Indications for UCBT were bone marrow failure in 191 patients (BMF, 93%) and acute leukemia/myelodysplasia in 14 patients (AL/MDS, 7%). Median age at transplant was 8.8 y (1.2-43) and 90% (n=185) were children <18y old. Graft sources included single (n=164), double (n=23) or UCB co-infused with other cell sources (n=18). Recipients and UCB pairs had 0-1/6 HLA mismatches in 48% (n=99). Most patients (n=196, 97%) received reduced intensity conditioning. Flu Cy-based (n=116; 57%) and Bu Cy ± Flu (n=48, 23%) were the most frequently used regimens. Total body irradiation (TBI) or total lymphoid irradiation (TLI) were administered to 28% (n=58) of patients at doses < 8 Gy. Serotherapy consisted of anti-thymocyte globulin (ATG, n=159; 78%) or alemtuzumab (n=8; 4%). Graft- vs-host disease (GVHD) prophylaxis was mainly based on CSA ± steroids (65%) or MMF ± steroids (32%). Median follow-up was 88 months (3-345). The cumulative incidence (CI) of neutrophil recovery was 79% (67-79) at day 60,with a median time of 20 (7-62) days (d). The 100d CI of grades II-IV acute GVHD (aGVHD) was 29.8% (24-37). The 5y CI of chronic GVHD (cGVHD) was 30.4% (24-38) with 12% extensive forms. The 1y-CI of non-relapse mortality (NRM) was 37.4% (31-45). At 5y follow-up, event-free survival (EFS) was54% ± 3 and overall survival (OS) was 55% ± 3. In multivariate analysis, HLA matching 0-1/6 (HR 2.0, p=0.01) and related UCBT (HR 2.4; p=0.001) were predictive of better engraftment. None of the tested factors had statistical significant impact on incidence of aGVHD or cGVHD. Negative recipient CMV serology (HR 0.29, p=0.003) and transplant year ≥2013 (HR 0.29; p=0.004) were the only factors associated with improved OS. Both factors were also predictive of better NRM and EFS. Fifty-one recipients of UCBT (3 related and 48 unrelated) experienced primary graft failure (GF). Twenty-six patients underwent subsequent allogeneic HCT within a median of 1.8 months (0.8 -6.8) after first UCBT. Seven subsequent HCT recipients (1 related bone marrow (BM), 1 related peripheral blood, 1 unrelated BM, and 4 unrelated UCB) were alive at last follow-up. Second neoplasms developed in 8 patients (4%) within a median interval of 9.7 y post-UCBT (3.6-13.5) and included one donor derived-AML and 7 solidtumors (1 skin, 2 upper GI, 4 oral cavity) . A totalof93 patients (45%) died from transplant related toxicity (n=85, including 42 who died after GF), second neoplasms (n=6) or unknown cause (n=2). Survival >2y was observed in 106 patients, but comprehensive data for late transplant complications were available only in 45 long-term survivors. For these 45, median post transplant follow-up was 8.7y (2.7-28.8). Late organ impairments ( table 1) included: endocrine disorders (32%), lung (24%), eyes (20%), kidneys (20%), immune system (18%), liver (16%), musculoskeletal, and bone (16%) diseases. Other dysfunctions involved: oral mucosa (11%), neurologic system (7%), gastrointestinal (4%), genital (13%) and urinary tracts (4%). Cardiovascular complications were rare (1 case of chronic pericarditis). Conclusion: Improved outcomes have been observed in recent years in FA patients receiving unrelated UCBT likely due to improved conditioning regimens, effective post-transplant care and better HLA parity. With longer survival, late multi-organ complications are observed. Identification of risk factors and life-long screening for early management of organ impairments and second neoplasms are mandatory to improve quality of life of transplant survivors and decrease late mortality.