Dose Of Cy Research Articles

Low‒Dose Cyclophosphamide Enhances the Tumoricidal Effects of 5-Day Spacing Stereotactic Ablative Radiotherapy by Boosting Antitumor Immunity.

To investigate the potential role of low‒dose cyclophosphamide (Cy) as a radiosensitizer by evaluating its impact on the immune response and the abscopal effect of stereotactic ablative radiotherapy (SABR) through preclinical models. CT26 tumors (immunologically hot) and 4T1 tumors (immunologically cold), grown in immunocompetent BALB/c and immunodeficient BALB/c‒nude mice, were irradiated with 20 Gy in two fractions with 5‒day spacing followed by intraperitoneal injections of 9 mg/kg Cy every 3 days. Immunological changes in CT26 tumors caused by the treatments were assessed using flow cytometry. Changes in the expression of HIF‒1α in tumors were also assessed. Splenocytes and bone marrow‒derived dendritic cells (DCs) were exposed to various concentrations of Cy to assess T cell proliferation and DC differentiation. The combination of Cy with RT (RT+Cy) significantly suppressed tumor growth compared to RT alone in immunocompetent mice, while that effect was not observed in immunodeficient mice. Additionally, RT+Cy effectively induced abscopal effects in hot and cold tumors, with increased CD8+ T cells in blood and tumors. Significantly higher expression levels of Granzyme B, IFN‒γ, and TNF‒α were observed in RT+Cy group compared to the RT alone group. In vitro data indicated that low‒dose Cy promotes DC differentiation. Low‒dose Cy suppressed the radiation‒induced upregulation of HIF‒1α in the tumors. Low‒dose Cy enhances tumoricidal effects of 5‒day spacing high‒dose RT by increasing anti-tumor immune responses.

Long-term survival after unrelated donor marrow transplantation for aplastic anaemia after optimized conditioning regimen: a retrospective multicentre cohort study

Almost all acquired severe aplastic anaemia is immune mediated and characterised by hypocellular bone marrow and ≥2 affected haematopoietic lineages. The optimal preparartive regimen for unrelated donor transplantation remains to be established. We aimed to study long-term outcomes after unrelated donor transplantation for severe aplastic anaemia with de-escalation of cyclophosphamide (Cy) dose in steps of 50mg/kg (150, 100, 50, 0mg/kg) in combination with total body irradiation (TBI) 2Gy, anti-thymocyte globulin (ATG) and fludarabine. Ninety-six patients with severe aplastic anaemia aged ≤65 years with adequate organ function enrolled on a trial of human leukocyte antigen (HLA)-matched or 1 HLA-locus mismatched unrelated donor marrow transplantation conducted between 02/2006 and 12/2013 in the United States (NCT00326417). Exclusion criteria were Karnofsky performance status of less than 60, clonal cytogenetic abnormalities and inherited marrow failure syndormes. The primary outcome was day-100 engraftment (achievement of absolute neutrophil recovery to at least 0.5×109/L without subsequent decline) and day-100 survival. The trial determined the lowest effective Cydose as 50mg/kg (n=38) for day-100 engraftment and survival. Cy dose 100mg/kg (n=41) was also acceptable. Accrual to Cy doses 150mg/kg (n=15) and 0mg/kg (n=3) was terminated early for toxicities. The current study is an extended follow up of patients enrolled on the trial (NCT00326477) and includes 76 of 96 patients alive ≥1 year after transplantation. There were 20 deaths in the first year after transplantation (Cy 0mg/kg [n=2], Cy 50mg/kg [n=1], Cy 100mg/kg [n=10], Cy 150mg/kg [n=7]). Patients were followed prospectively from transplantation and data reported using standardized data collection forms until death, loss to follow up or last contact through November 2023. The incidence of graft failure was calculated using the cumulative incidence estimator and the probability of survival using the Kaplan-Meier estimator. The median follow up of the cohort is 8.02 (IQR) 5.16-10.12) years. With Cy 50mg/kg, there was one graft failure and five deaths ≥1 year after transplantation. With Cy 100mg/kg there was only one late death and no graft failure. The 8-year probabilities of survival were 85.0% (95% CI 67.3-93.5) and 75.6% (95% CI 59.4-86.1) after Cy50mg/kg and 100mg/kg, respectively, P=0.31. With Cy 0mg/kg and 150mg/kg, there were no graft failures or death ≥1 year after transplantation. Regardless of Cy dose 12 of 15 patients aged ≥50 years died. Cy 50mg/kg or 100mg/kg with TBI 2Gy, ATG and fludarabine are effective conditioning regimens for unrelated donor marrow transplants for aplastic anaemia. Identification of an optimized transplantation approach for patients aged ≥50 years is needed. US National Institutes of Health.

Efficacy of a Modified Post-Transplant Cyclophosphamide Regimen for Unrelated Donor Hematopoietic Stem Cell Transplantation in Patients with Severe Aplastic Anemia: A Prospective Study

The aim of the present study was to examine the efficacy of the modified post-transplant cyclophosphamide (PTCy) regimen, which involved reducing the Cy dose to 40 mg on days +3 and +4 in patients with severe aplastic anemia (SAA) subjected to unrelated donor allogeneic hematopoietic stem cell transplantation (URD-HSCT). For this purpose, a prospective single-center trial was conducted and the clinical outcomes were collected from 30 patients with SAA treated with the modified PTCy regimen for URD-HSCT. The median time to neutrophil and platelet engraftment was 13 days (range, 11 to 16) and 12 days (range, 5 to 33), respectively. The cumulative incidence of neutrophil and platelet engraftment was 93.1% ± 0.3% and 96.6% ± 0.2%, respectively. The 2-year overall survival (OS) was 97% (95% confidence interval [CI]: 90%-100%] and 2-year graft-versus-host disease (GVHD) and rejection-free survival (GRFS) was 93% (95% CI: 85%-100%). The incidence rates of acute GVHD (aGVHD) and chronic GVHD (cGVHD) were 13.8 ± 0.4% and 10.3 ± 0.3%, respectively, and no patients developed grades III-IV aGVHD. However, only one patient developed a moderate extensive cGVHD. The incidence of reconstitution varies among different subsets of immune cells after URD-HSCT. Natural killer (NK) cells recover first, followed by CD8+ T and CD19+ B cells, and finally CD4+ T cells. In conclusion, the present study demonstrates that the modified PTCy regimen, with a reduced dose of 40 mg on days +3 and +4, may be an effective regimen for URD-HSCT in patients with SAA and reduce the occurrence of the GVHD.

Abstract CT070: Solid tumor patients require higher cyclophosphamide (cy) dose than multiple myeloma (MM) patients to achieve adequate lymphodepletion (LD) necessary to enable allogeneic CAR-T expansion

Abstract Introduction: Poseida is developing innovative allogeneic T stem cell memory rich CAR-T both for hematologic malignancies (HM) and solid tumors (ST) including P-BCMA-ALLO1 which targets BCMA for MM, and P-MUC1C-ALLO1 targeting MUC1-C for epithelial-derived ST. Optimal LD for allogeneic cell therapies remains to be established. Most research has focused on HM, where patients (pts) have likely undergone HSCT and are, therefore, LD experienced in contrast to ST pts. Consequently, ST pts may require higher doses of LD chemotherapy to achieve LD depth comparable to HM pts. This study sought to compare LD with higher doses of cy across our early phase 1 trials (NCT04960579/NCT05239143) which are enrolling ST and MM pts, respectively. Methods: P-BCMA-ALLO1 and P-MUC1C-ALLO1 are 3+3 phase 1 dose escalation studies enrolling heavily pre-treated pts. LD arm S used cy 300 mg/m2 + fludarabine (flu) 30 mg/m2 × 3 days, followed by CAR-T. Alternative arms tested higher cy doses: P1 (500 mg/m2) and P2 (1,000 mg/m2), both with flu. We assessed LD depth and cellular kinetics (CK) by measuring WBC, serum IL-15, and CAR-T transcripts (qPCR). Results: By Jan 4, 2024, 42 pts received P-BCMA-ALLO1 (22 in S, 13 in P1, 7 in P2), and 30 pts received P-MUC1C-ALLO1 (19 in S, 9 in P1, 2 in P2). ST pts presented with higher baseline WBC compared to MM pts with median counts of 5.7×103 vs. 4.4×103/µL. LD was less pronounced in ST pts with a median WBC nadir of 1.1×103/µL for both S and P1. In MM pts median WBC nadir was 0.6×103 for S vs. 0.2×103 for P1. For P-BCMA-ALLO1, CK at 2×106 cells/kg dose showed a mean Cmax of 130,811 copies (cp)/µg in P2 and 6,191 in P1 compared to 170 in S. Median WBC nadir was significantly (p=0.0002) lower for P2 (0.1×103/µL) compared to S (0.6×103), with corresponding AUC0-13d values of 3.5×103/µL for P2 vs. 10×103 for S (p=0.0013). Peak serum IL-15 in P2 was 66.1 pg/mL, significantly higher than 29.7 in S (p=0.0110). In P1, there was a trend towards improved LD with median WBC nadir for P1 of 0.3×103/µL and AUC0-13d of 6.4×103/µL.P1 LD for P-MUC1C-ALLO1 did not improve CK, with WBC nadir and AUC 0-13d values comparable to S. P-MUC1C-ALLO1 CK at 2×106 cells/kg showed a mean Cmax of 692 cp/µg DNA in S, 23 in P1, and 4,200 in P2. P-MUC1C-ALLO1 pts receiving P2 achieved the deepest LD (WBC nadir: 0.1×103/µL). Peak IL-15 in ST pts was increased and delayed to day 8 in P2, compared to day 0 - 1 in S or P1. Conclusion: Increasing cy dose to 500 or 1,000 mg/m2 improves CK for P-BCMA-ALLO1 pts due to enhanced LD depth. For P-MUC1C-ALLO1 pts, increasing cy to 500 does not improve LD depth or CK compared to 300. This difference suggests the need for higher LD doses in ST pts than MM pts to achieve optimal CK. This differential need for LD chemo for these two studies is unlikely due to cellular characteristics because of similar manufacturing technology with the only difference being the binder. Citation Format: Sabrina Haag, Jeff D. Eskew, Katherine McArthur, Joanne McCaigue, Sepideh Vaziri, Samuel DePrimo, Christopher E. Martin, Catherine Gregovics, Ann Murphy, Hamid Namini, Ellen Christie, Marcela Marinez-Prieto, Rajesh Belani, Stacey Cranert, Julia Coronella, Devon Shedlock. Solid tumor patients require higher cyclophosphamide (cy) dose than multiple myeloma (MM) patients to achieve adequate lymphodepletion (LD) necessary to enable allogeneic CAR-T expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT070.

Lymphodepletion - an essential but undervalued part of the chimeric antigen receptor T-cell therapy cycle.

Lymphodepletion (LD) or conditioning is an essential step in the application of currently used autologous and allogeneic chimeric antigen receptor T-cell (CAR-T) therapies as it maximizes engraftment, efficacy and long-term survival of CAR-T. Its main modes of action are the depletion and modulation of endogenous lymphocytes, conditioning of the microenvironment for improved CAR-T expansion and persistence, and reduction of tumor load. However, most LD regimens provide a broad and fairly unspecific suppression of T-cells as well as other hematopoietic cells, which can also lead to severe side effects, particularly infections. We reviewed 1271 published studies (2011-2023) with regard to current LD strategies for approved anti-CD19 CAR-T products for large B cell lymphoma (LBCL). Fludarabine (Flu) and cyclophosphamide (Cy) (alone or in combination) were the most commonly used agents. A large number of different schemes and combinations have been reported. In the respective schemes, doses of Flu and Cy (range 75-120mg/m2 and 750-1.500mg/m2) and wash out times (range 2-5 days) differed substantially. Furthermore, combinations with other agents such as bendamustine (benda), busulfan or alemtuzumab (for allogeneic CAR-T) were described. This diversity creates a challenge but also an opportunity to investigate the impact of LD on cellular kinetics and clinical outcomes of CAR-T. Only 21 studies explicitly investigated in more detail the influence of LD on safety and efficacy. As Flu and Cy can potentially impact both the in vivo activity and toxicity of CAR-T, a more detailed analysis of LD outcomes will be needed before we are able to fully assess its impact on different T-cell subsets within the CAR-T product. The T2EVOLVE consortium propagates a strategic investigation of LD protocols for the development of optimized conditioning regimens.

Effects of Cyfluthrin Exposure on Neurobehaviour, Hippocampal Tissue and Synaptic Plasticity in Wistar Rats.

This experiment was conducted to study the effects of Cyfluthrin (Cy) exposure on neurobehaviour, hippocampal tissue and synaptic plasticity in Wistar rats. First, it was found that high-dose Cy exposure could cause nerve injury, resulting in symptoms such as deficits in learning and memory ability, spatial exploration and autonomic motor function. Moreover, it was found that medium- and high-dose Cy exposure could cause an abnormal release of the neurotransmitter Glu. Second, brain tissue pathology showed that the middle and high doses of Cy caused tissue deformation, reduced the number of hippocampal puramidal cells, caused a disorder of these cells, decreased the number of Nissl bodies, and caused pyknosis of the hippocampal cell nuclear membrane and serious damage to organelles, indicating that exposure to these doses of Cy may cause hippocampal tissue damage in rats. Third, as the exposure dose increased, morphological changes in hippocampal synapses, including blurred synaptic spaces, a decreased number of synaptic vesicles and a decreased number of synapses, became more obvious. Moreover, the expression levels of the key synaptic proteins PSD-95 and SYP also decreased in a dose-dependent manner, indicating obvious synaptic damage. Finally, the study found that medium and high doses of Cy could upregulate the expression of A2AR in the hippocampus and that the expression levels of inflammatory factors and apoptosis-related proteins increased in a dose-dependent manner. Moreover, the expression of A2AR mRNA was correlated with neurobehavioural indicators and the levels of inflammatory factors, synaptic plasticity-related factors and apoptosis-related factors, suggesting that Cy may cause nerve damage in rats and that this effect is closely related to A2AR.

Long-Term Outcomes after Unrelated Marrow Transplantation for Aplastic Anemia with Optimized Cyclophosphamide Dose (BMT CTN 0301)

Background: Seventy-nine patients (aged ≤65 years) with aplastic anemia received HLA-matched or single locus mismatched unrelated donor marrow transplantation (BMT) on a clinical trial ( BMT CTN 0301; NCT00326417) between 02/2006 and 12/2013 ( Lancet Haematol. 2015, 2:e367-75). The median ages of patients enrolled on the cyclophosphamide (Cy) 50 mg/kg and 100 mg/kg strata were 25 and 18 years, respectively, and 80% were Caucasians. That trial determined the minimum dose of Cy required for engraftment when added to total body irradiation (TBI 2 Gy), anti-thymocyte globulin (rabbit-ATG 9 mg/kg) and fludarabine (120 mg/m 2). Graft versus host disease (GVHD) prophylaxis was cyclosporine and methotrexate. Although Cy at 50 mg/kg (n=38) and 100 mg/kg (n=41) were both effective for day 100 and 1-year outcomes, the computed optimum Cy dose by Bayesian method was 50 mg/kg. Early results showed a 1-year incidence of graft failure after Cy 50 mg/kg and 100 mg/kg of 12% and 15%, respectively. The corresponding probabilities of 1-year survival were 92% and 76%. Methods: Thirty-seven patients who received Cy at 50 mg/kg and 31 patients who received Cy at 100 mg/kg were alive ≥1 year after BMT. These patients now have median follow ups of 7 and 8.7 years, respectively. The incidence of graft failure and chronic GVHD was calculated using the cumulative incidence estimator and the probability of survival using the Kaplan-Meier estimator. Results: In the trial cohort, 31 of 38 patients (82%) who received Cy 50 mg/kg were HLA-matched to their donor at HLA-A, B, C and DRB1, compared to 27 of 41 (66%) receiving Cy 100 mg/kg. Table 1 shows outcomes of the 38 and 41 patients who received Cy at 50 mg/kg and Cy at 100 mg/kg. With Cy 50 mg/kg there was 1 late graft failure at 4 years (cumulative total 5). Five deaths occurred at 1.5, 1.8, 4.2, 5.2 and 12.2 years after BMT (cumulative total 6). All chronic GVHD occurred within 2 years. Late toxicities (n=8; 22%) included liver cirrhosis (n=1, 1.25 years), myocardial infarction (n=1, 3.8 years), congestive heart failure (n=1, 8.7 years), acute renal failure requiring dialysis (n=1, 3.8 years), avascular necrosis (n=1, 9 years), gonadal dysfunction in pre-pubertal children at BMT (n=2, 1.4 and 3.9 years), and secondary cancer (n=1, pancreatic adenocarcinoma 3.1 years). The 8-year survival among the 38 patients receiving Cy 50 mg/kg was 85% despite late toxicities, including death, being more common beyond the first year after BMT. With Cy 100 mg/kg, there were no late graft failures. There was 1 death at 8.6 years (in a patient with underlying dyskeratosis congenita (cumulative total 11). One patient was diagnosed with chronic GVHD at 7 years. Late toxicities (n=6; 19%) included pulmonary (n=2, ARDS 8 years and restrictive airway disease 1.9 years), chronic renal failure requiring dialysis (n=1, 15 years), avascular necrosis (n=1, 1.5 years), gonadal dysfunction (n=1, 2.6 years), and secondary cancer (n=1, myelodysplastic syndrome 8.9 years). The 8-year survival among the 41 patients receiving Cy 100 mg/kg was 76% and all except 1 death occurred within the 1 st year after BMT. Causes of death after the first year of BMT are shown in Table 2. Conclusion: Eight-year survival ≥75% and sustained engraftment confirm both Cy at 50 mg/kg and Cy at 100 mg/kg with TBI 2 Gy, ATG and fludarabine are effective conditioning for unrelated BMT for aplastic anemia but life-long surveillance for late complications is required. Chronic GVHD was common and proved fatal in several patients despite most donor-recipient pairs who received Cy at 50 mg/kg being HLA-matched at the allele-level.

Intensity of Cyclophosphamide-Based Bridging Therapy Before Chimeric Antigen Receptor T Cell Therapy in Myeloma

Patients receiving autologous chimeric antigen receptor T-cell (CAR-T) therapies for multiple myeloma (MM) may require bridging therapy (BT) before CAR-T infusion to maintain some level of disease control. Alkylators such as cyclophosphamide (Cy) are often used as parts of BT regimens, either in high-intensity regimens such as modified hyperCVAD or once-weekly regimens such as KCd. However, there is no consensus around the optimal BT alkylator dose intensity in MM. We performed a single-center analysis of all instances of BT before planned autologous CAR-T for MM during a 5-year period ending April 2022. We classified bridging regimens into three cohorts: (1) hyperfractionated Cy (HyperCy) with inpatient Cy every 12-24 hours or as continuous intravenous infusions, (2) less intensive Cy dosing (WeeklyCy) such as KPd or KCd, and (3) NonCy, where no alkylators were used in BT. Demographic, disease-related, and treatment-related characteristics were collected for all patients. The three BT cohorts were compared using Fisher's exact tests, Kruskal-Wallis tests, and log-rank tests where appropriate. We identified 70 discrete BT instances among 64 unique patients: 29 (41%) with HyperCy, 23 (33%) with WeeklyCy, and 18 (26%) with NonCy. Median total levels of Cy dosing during BT were 2100 mg/m2, 615 mg/m2, and 0 mg/m2 respectively. Age, number of prior lines, triple-class refractory status, presence of high-risk cytogenetics, extramedullary disease, bone marrow plasma cell burden, involved free light chain (iFLC) kinetics before collection, and other measures of disease aggressiveness were comparable between cohorts. iFLC levels rose ≥25% and ≥100 mg/L during BT (approximating progressive disease) in comparable proportions (p=0.25) between cohorts: 52% HyperCy, 39% WeeklyCy, and 28% NonCy. All BT instances without subsequent CAR-T were due to manufacturing failures. Among 61 instances of bridging followed by CAR-T, vein-to-vein times were slightly longer (p = 0.03) with HyperCy (45 days) vs WeeklyCy (39 days) and NonCy (46.5 days). Neutrophil recovery timeframes were similar between cohorts, but platelet recovery took longer with HyperCy (64 days) vs WeeklyCy (42 days) and NonCy (12 days). PFS was comparable between cohorts, but median OS was not: 15.3 months with HyperCy versus 30.0 months with WeeklyCy and not reached with NonCy. In our retrospective analysis of BT before CAR-T therapy in MM, HyperCy did not result in superior disease control than WeeklyCy despite a threefold higher dose of Cy. In contrast, HyperCy was associated with longer post-CAR-T platelet recovery and worsened OS despite comparable measurements of disease aggressiveness and tumor burden. Study limitations include our small sample size as well as confounding from gestalt markers of MM aggressiveness that might have led both to poorer outcomes as well as physicians' decisions to prescribe HyperCy. Given the rarity of objective disease responses to chemotherapy in relapsed/refractory MM, our analysis suggests that hyperfractionated Cy regimens do not outperform once-weekly Cy regimens for most patients who require BT before CAR-T therapy.

Alternative Bone Marrow Transplantation with Post-Transplant Cyclophosphamide (PTCy) As Initial Therapy for Acquired Severe Aplastic Anemia (SAA)

Immunosuppressive therapy (IST) is standard front-line treatment for SAA, except for patients aged <20 years with a suitable HLA-matched sibling donor (MSD) for BMT. The hematopoietic response after IST, even with the addition of eltrombopag, is ~ 70%-80% and 5-year survival is 60% to 85%. Failure-free survival (alive and in remission without clonal disease) beyond 10 years after IST is ~50%. In contrast, long-term survival after BMT is ~90% in patients aged < 20 years, and 75% in older patients. Currently, BMT with an unrelated or HLA-haploidentical (haplo) donor is reserved for failure of IST because of morbidity and mortality concerns. PTCy improves the safety and efficacy of haplo BMT by facilitating engraftment and decreasing graft-vs-host disease (GVHD), with survival comparable to MSD BMT. Here we review the outcomes of alternative donor BMT and PTCy in treatment-naïve (TN) SAA patients. Eligible patients met criteria for SAA and were TN. Fanconi anemia and telomere biology disorder patients were excluded. Non-myeloablative (NM) conditioning included rabbit antithymocyte globulin (0.5 mg/kg day-9 and 2 mg/kg on days -8,-7), fludarabine (30mg/m2/day on days -6 to -2), low dose CY (14.5 mg/kg on days -6, -5) and total body irradiation (TBI) (200cGy or 400 cGy day -1). After the marrow graft was infused on day 0, cyclophosphamide 50 mg/kg/day IV was administered on days 3 and 4 post-transplant. All received mycophenolate mofetil (MMF) from day 5-35 and tacrolimus from day 5 through 1 year. Neutrophil recovery was defined as occurring on the first of three consecutive days of an ANC >1.0 x109/L. Red cell recovery was defined as days from transplant to the last red blood cell transfusion. Platelet recovery was defined as the first day of a platelet count >50,000 without transfusion in the preceding 7 days. Thirty-two eligible patients were treated at Johns Hopkins between 9/2016 and 5/2022. Median follow-up for all patients is 40 months (range 2-78). Table 1 shows demographics. The median age was 25 years (range 3-63) with 24 patients age >20 years, and 56% were males. Median time to neutrophil recovery was 20 days (range 14-88). Median time to red cell recovery was 25 days with 90% transfusion independence by day 60. Median time to platelet recovery was 25 days with 90% transfusion independence by day 100. TBI dose was increased from 200 to 400 cGy to reduce graft failure after graft loss in 3 of the first 7 patients; 2 of these 3 died (from infection and graft failure) and one has been re-transplanted and is now 100% donor. Overall survival at 3 years post-transplant is 93%. (Figure 1) All 24 consecutive patients who received 400 cGy are alive and well with engraftment. CMV reactivated in 13 patients, and 6 required therapy. Rates of acute and chronic GVHD were both 7%, none developed grade 3-4 acute or extensive chronic GVHD, and all patients beyond day 365 are off treatment for GVHD. No secondary malignancies have been observed. NM BMT with PTCy, using mostly haplo donors, can cure SAA. Increasing TBI dose from 200 to 400 cGy improves engraftment. Rates of infection and mortality are not increased compared to standard upfront IST. Historical data show that >50% of patients in this age range require transplantation subsequent to IST. Upfront BMT, as described here, allows patients to come off immunosuppression by 6-12 months post-BMT, prevents the morbidity of prolonged IST, and may reduce healthcare costs. Most patients receiving 200 cGy TBI maintain fertility; we are monitoring fertility rates after 400cGy. This approach forms the basis for the upcoming Bone Marrow Transplant Clinical Trials Network multicenter study in TN acquired SAA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cyclophosphamide, hydroxycyclophosphamide and carboxyethyl phosphoramide mustard quantification with liquid chromatography mass spectrometry in a single run human plasma samples: A rapid and sensitive method development

A novel method for simultaneous quantification of cyclophosphamide along with its two major metabolites namely 4-hydroxycyclophosphamide (HCy) and carboxyethyl phosphoramide mustard (CEPM) in a single sample run was demonstrated in the present study. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument was used for analysis. Semicarbazide was used as a stabilizing agent for HCy whereas ifosfamide, hexamethyl phosphoramide mustard and deuterated CEPM were the internal standards for quantification of Cy, HCy and CEPM respectively. Chromatographic separation was achieved by Chromsystems C18 reverse-phase column (50 mm × 4.6 mm, particle size 3.2 µm). The mobile phase was composed of eluent A (2 mM ammonium acetate in water with 2% formic acid) and eluent B (100 % acetonitrile). The flow rate was 1 ml/min. Linearity of the assay was assured in the range of 19.53 ng/ml to 10,000 ng/ml concentration in human plasma, which is adequate for pharmacokinetic studies of any dose Cy used clinically. The quality control(QC) accuracy was between 99.58% and 101.62%, 97.85% to 103.53% and 99.64% to 100.10% for Cy, HCy and CEPM respectively. Precision limits for QC samples were between 3.9% and 9.4%, 5.2% to 8.9% and 1.8% to 9.2% respectively. The analytical method was validated in ten leukaemia patients undergoing haploidentical hematopoietic cell transplantation.

Single Cord Blood Transplantation Versus HLA-Haploidentical Related Donor Transplantation Using Post-Transplant Cyclophosphamide in Patients with Hematological Malignancies

Introduction: Unrelated cord blood (UCB) and haploidentical related donor transplantation using post-transplant cyclophosphamide (PTCy-haplo) have become alternative options to treat patients with hematological malignancies without a human leukocyte antigen (HLA)-matched donor. Although a previous phase III study showed better outcomes after bone marrow transplantation (BMT) using PTCy-haplo than those after double unit UCB, a donor selection algorithm including peripheral blood stem cell transplant (PBSCT) using PTCy-haplo or high-risk patients remains to be clarified. With the aim to assess the relative efficacies of UCB and PTCy-haplo transplant, we performed a multi-center retrospective analysis to compare the clinical outcomes in patients with hematologic malignancies.Methods: We included 517 patients aged 16 years to 70 years with hematological malignancies who received a first stem cell transplantation using a single UCB unit or PTCy-haplo between 2013 and 2019 in Kyoto Stem Cell Transplantation Group (KSCTG), which is a multi-center group of 18 transplantation centers in Japan. The primary endpoint was to compare overall survival (OS) between the UCB and PTCy-haplo groups. Secondary endpoints were relapse-free survival (RFS), GVHD- and relapse-free survival (GRFS), relapse, non-relapse mortality (NRM), neutrophil engraftment, platelet engraftment, grade II-IV acute graft-versus-host disease (GVHD), grade III-IV acute GVHD, chronic GVHD, and extensive chronic GVHD.Results: Among 517 patients, 460 received single UCB transplant and 57 received PTCy-haplo related donor transplant (53 PBSCT and 4 BMT). The median age of the UCB and PTCy-haplo groups was 55 and 52 years, respectively (P= 0.770). Myeloablative conditioning (MAC) was used more often in the UCB group than in the PTCy-haplo group (P< 0.001). In the PTCy-haplo cohort, the total dose of Cy was 80mg/m 2 (43.9%) or 100mg/m 2 (56.1%), and the median duration of tacrolimus and mycophenolate mofetil use was 173 and 34 days, respectively. The median follow-up periods of survivors were 2.2 and 2.4 years, respectively. OS in the UCB group was comparable to that in the PTCy-haplo group (2-year OS; 53.7%, 53.6%, P= 0.71 and adjusted hazard ratio (aHR) 1.00, P= 0.99), with similar risks of relapse (aHR 0.91, P= 0.99) and NRM (aHR 0.93, P= 0.69). This result was consistent regardless of disease risk. Neutrophil and platelet engraftment were significantly lower (92.2% vs 94.7%, P= 0.042 and 79.8% vs 82.5%, P= 0.003) and the incidence of acute GVHD in the UCB group tended to be higher (gradeⅡ-Ⅳ; aHR 1.64, p= 0.055, grade III-IV; aHR 3.60, P= 0.073) in the UCB group than those in the PTCy-haplo group. However, the incidences of chronic or extensive chronic GVHD after UCB transplant were comparable (chronic; aHR 0.89, p=0.68, extensive chronic; aHR 0.69, P= 0.35) to those after PTCy-haplo transplant. In the subgroup analysis of disease-specific comparison, acute leukemia patients have a significantly lower risk of relapse with UCB transplant (UBC vs PTCy-haplo, HR 0.55, P= 0.030) and lymphoma patients tended to have good results after PTCy-haplo transplant in terms of higher OS (UCB vs PTCy-haplo, aHR 2.13, P= 0.162) and lower relapse (aHR 3.43, P= 0.100). In terms of Cy toxicity in the PTCy-haplo group, transplant with a reduced dose of Cy (80 mg/m 2) tended to show higher OS (2-year OS, 64.8% vs 45.0%) and significantly lower NRM (2-year NRM, 8.0% vs 31.2%) without an increase in gradeⅡ-IV acute GVHD compared to those after standard-dose Cy (100 mg/m 2).Conclusion: Our findings suggest that UCB transplant gives outcomes comparable to PTCy-haplo transplant for patients without an HLA-matched sibling or unrelated donor. DisclosuresKanda: Amgen Astellas BioPharma: Honoraria; Astellas Pharma Inc.: Consultancy, Honoraria; Bristol-Myers Squibb Co: Honoraria; CHUGAI PHARMACEUTICAL Co., Ltd.: Honoraria; DAIICHI SANKYO Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Eisai: Research Funding; Janssen Pharmaceutical K.K.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin Co., Ltd.: Honoraria; Megakaryon Co: Honoraria, Membership on an entity's Board of Directors or advisory committees; NextGeM Inc: Patents & Royalties; Novartis Pharma K.K.: Honoraria; Ono Pharma Inc.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Sanofi K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; SymBio Pharmaceuticals, Ltd.: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Company Limited: Honoraria, Membership on an entity's Board of Directors or advisory committees; TEIJIN PHARMA LIMITED.: Honoraria. Imada: Celgene Co., Ltd.: Honoraria; Bristol-Myers Squibb K.K.: Honoraria; Astellas Pharma Inc.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; Otsuka Pharmaceutical Co. Ltd.: Honoraria; Takeda Pharmaceutical Co. Ltd.: Honoraria; Novartis Pharma K.K.: Honoraria. Kondo: Asahi Kasei Pharmaceutical: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria; Chugai Pharma: Honoraria; MSD Pharmaceutical: Honoraria; Sumitomo Dainippon Pharma: Honoraria. Takaori-Kondo: Bristol-Myers K.K.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Research Funding; Celgene: Research Funding.

N-Acetylcysteine Amide Alleviates Cyclophosphamide-Induced Endothelial Damage

Rui He 1,2, Wenyi Zheng 1,2, Terra Slof 3, Eva Wärdell 4, Agneta Månsson-Broberg 4, Svante Norgren 5, Per Ljungman 2, Ying Zhao 1, 2 , Moustapha Hassan 1, 21.Experimental Cancer Medicine, Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, 141 86 Huddinge, Sweden2.Center of Allogeneic Stem Cell Transplantation (CAST) and Clinical Research Center, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden.3.Institute for Life Sciences and Chemistry, HU University of Applied Sciences Utrecht, Oudenoord 330, 3513 EX Utrecht, Netherlands4.Department of Medicine, Division of Cardiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.5.Children´s and Women´s Health Theme, Karolinska University Hospital and Karolinska Institutet, Stockholm, SwedenAimThe current study aimed to investigate the protective role of the novel antioxidant N-acetylcysteine amide (NACA) in cyclophosphamide (Cy)-induced endothelial injury and to explore the underlying mechanism.BackgroundThe alkylating agent Cy is still one of the important corner stones in cancer treatment. Cy is used also as a part of the conditioning regimens prior to hematopoietic cell transplantation (HCT) and as a prophylactic treatment post transplantation in graft-versus-host disease (GVHD). Several studies have shown that high doses of Cy are associated with a number of adverse effects including alopecia, gonadal toxicity, hemorrhagic cystitis, carcinogenesis and late cardiovascular toxicity. Moreover, we have shown that treatment with high-dose Cy caused damage to the arterial endothelium, which might contribute to late cardiovascular disorders. Hitherto, there is no prophylactic strategy for Cy-induced endothelial toxicity. Oxidative stress is one of the main players in the pathogenesis of endothelial disorders and hence is an exploitable trait for targeted treatment.MethodsThe prophylactic efficacy of NACA was evaluated using human aortic endothelial cells (HAECs) and human umbilical endothelial cells (HUVECs). The efficacy of NACA was compared with the clinically approved derivative N-acetylcysteine (NAC). Cy is a prodrug that cannot be activated in the endothelial cells. Therefore, cells were incubated with Cy active metabolite, 4-hydroxycyclophosphamide (4-OH-Cy). In order to reveal the underling mechanism of action, we have investigated and evaluated the following parameters: alteration in cell morphology, cell death pathway, inhibition of DNA damage, variation of redox hemostasis, and neovascularization.ResultsWe found that pre-incubation with 5 mM NACA preserved the normal endothelial cell morphology. However, for the quantification of the obtained results, cell colonies were visualized using crystal violet staining while cell viability was determined using WST-1 assay. Moreover, NACA was found to decrease 4-OH-Cy-induced DNA damage and cell death via caspase-dependent apoptosis in both HAECs and HUVECs. In addition, NACA significantly inhibited 4-OH-Cy-induced oxidative stress by reducing the intracellular levels of reactive oxygen species (ROS) and by improving the cellular antioxidative capacity. Increased activities of catalase and superoxide dismutase (SOD) together with increased levels of reduced glutathione (GSH) were observed. Meanwhile, NACA pre-treatment rebalanced endothelial nitric oxide synthase (eNOS) and arginase I in HAECs, suggesting that NACA may be used as prophylactic strategy to avoid the impaired endothelium-dependent vasodilation caused by 4-OH-Cy. Moreover, data from tube formation assay indicated that NACA preserved the angiogenic capability of endothelial cells that was compromised by 4-OH-Cy through blockage of Notch signaling pathway. Our results showed that in contrast to NAC, its amide derivative NACA showed remarkably superior ability in alleviating the endothelial damage caused by 4-OH-Cy.ConclusionsIn summary, the present investigation showed that the novel antioxidant NACA could be applied to protect against 4-OH-Cy-induced endothelial cell dysfunction and death. Higher bioavailability and better cellular uptake make NACA to a better prophylactic agent compared to NAC. To translate these findings into the clinic, additional in vivo studies are warranted to confirm the protective potential of NACA. DisclosuresLjungman: Takeda: Consultancy, Other: Endpoint committee, speaker; Janssen: Other: Investigator; OctaPharma: Other: DSMB; Enanta: Other: DSMB; Merck: Other: Investigator, speaker; AiCuris: Consultancy.

Pomalidomide (Pom) Dexamethasone (D) With Or Without Oral Weekly Cyclophosphamide (Cy) For Lenalidomide Refractory Multiple Myeloma (LRMM): A Multicenter Randomized Phase II Trial

IntroductionPomD results in an overall response rate of 30% and median PFS of 3.8 months in patients with prior lenalidomide (L) and bortezomib (Vij et al. ASCO 2012). We thought to investigate the addition of oral weekly Cy to PomD in patients with LRMM. We have previously reported that the recommended phase II dose of Cy with standard dose Pom D was 400 mg PO D1, 8, 15. We herein report aggregated results of the phase I/II trial. PatientsEligible patients had relapsed and refractory myeloma after at least 2 prior therapies and were L refractory. Patients had a platelet count≥50,000/ mm3 and ANC≥1,000/mm3 (patients with ≥50% bone marrow plasmacytosis were allowed if platelet count ≥30,000/mm3 and ANC could be supported with GCSF during screening and therapy). In the phase II portion, patients were randomized (1:1) to receive pomalidomide 4mg PO D1-21, dexamethasone 40 mg PO D1,8,15,22 (20mg if older than 75 years) with or without oral cyclophosphamide 400 mg PO D1, 8, 15 of a 28 days cycle. Patients randomized to PomD were allowed to cross over to the PomCyD in the event of disease progression. Thromboprophylaxis was mandated with aspirin, warfarin or LMWH. Responses were assessed per the IMWG criteria with the addition of minimal response (MR, ≥25% decrease in serum component). ResultsBetween 1/2012 and 7/2013, 70 patients were enrolled (10 in the phase I portion) and 30 patients were randomized to the PomCyD and 30 to PomD. The median age was 65 years (range 44-80) and 41 (58%) were male. The median number of prior therapies was 5 (range 2-14). All patients were lenalidomide refractory and 55 (79%) were refractory to proteasome inhibitors. 19 patients (27%) had high risk cytogenetics (del 17p, t(4;14), t(14;16)) of which 12 patients (17%) had del 17p. In addition, 15 (21%) had 1q21 trisomy or tetrasomy. Aggregated responses are reported for the cohort. Of the 66 patients evaluable for response, 1 achieved a CR, 10 achieved a VGPR (15%), 21 achieved a PR (32%), 13 achieved a MR (20%) and 11 achieved SD (16%). The overall response rate (PR and better) was 48.5% and the clinical benefit rate (MR and better) was 68.2%. The median progression free survival (PFS) was 6.4 months (95% CI 2.6-10.2). Patients with ≥PR had a median duration of response of 10.4 months (95% CI 6.1-14.7). The most common grade ¾ adverse event regardless of attribution were neutropenia (30% of patients), febrile neutropenia (13%), pneumonia (13%), thrombocytopenia (11%) while grade 3/4 thromboembolic events were noted in 4%. ConclusionThe aggregate response rates (≥PR in 48%) and PFS of this all oral regimen compare favorably with published results using PomD in LRMM. Enrollment on the randomized phase II trial continues. Per protocol and to avoid alpha-spending, non-aggregated results of the two arms will be presented at the meeting. Disclosures:Baz:Sanofi: Research Funding; Karyopharm: Research Funding; Novartis: Research Funding; Bristol Myers Squibb: Research Funding; Millenium: Research Funding; Celgene: Research Funding. Off Label Use: pomalidomide dexamethasone in combination with cyclophosphamide for relapsed refractory myeloma. Alsina:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Shain:Celgene: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding. Wolf:Celgene: Honoraria, Research Funding; Millenium: Honoraria; Onyx: Honoraria. Mahindra:millenium: Membership on an entity's Board of Directors or advisory committees. Chari:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees. Jagannath:Millennium: Honoraria; Celgene: Honoraria.

Post Transplantation Cyclophosphamide Improves Outcome of Autologous Hematopoietic Stem Cell Transplantation in Animal Model of Multiple Sclerosis

Experimental allergic encephalomyelitis (EAE) is the animal model of multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (AHSCT) has recently been recognized as the standard treatment for MS. The aim of our experiment was to investigate the effect of AHSCT with the addition of low-dose post-transplantation cyclophosphamide (Cy) on EAE in rats. Low dose post-transplantation Cy is used in haploidentical HSCT to reduce the risk of graft versus host disease. We hypothesized that it could bring additional benefit in autologous HSCT in autoimmune diseases. Rats with evoked EAE were treated with high dose (125 mg/kg) Cy, followed by AHSCT or high dose (125 mg/kg) Cy followed by AHSCT followed by low dose (20 mg/kg) Cy in two-time schedules—with the therapy applied during the pre-symptomatic or symptomatic phase of the disease. Both AHSCT and AHSCT with post-transplantation Cy in accordance with both time schedules reduce the intensity of the inflammatory response in the CNS, in comparison with non-treated EAE rats. The reduction of clinical symptoms was present in all AHSCT treatment protocols, however, it was significantly stronger when post-transplantation Cy was given during the symptomatic phase of the disease. AHSCT with the addition of post HSCT low dose Cy improved the results of AHSCT by not only reducing the intensity of inflammation in the CNS but also by significantly reducing the clinical symptoms in treated animals when compared to AHSCT alone. We provide an experimental rationale that the addition of post-transplantation Cy may improve the outcome of HSCT in MS.Graphic

Non-TBI Based Conditioning Regimen in Peripheral Blood Stem Cell Transplantation (PBSCT) for Adult Acute Lymphoblastic Leukemia Patients: A Single Center Experience

Introduction: The outcome of adult acute lymphoblastic leukemia (ALL) patients with chemotherapy only is not promising and overall survival (OS) does not exceed 30%. Allogeneic Hematopoietic Stem Cell transplantation (allo-HSCT) in first or beyond complete remission (≥CR1) has shown a superior OS rate of approximately 50 % over conventional chemotherapy protocols. Total body irradiation plus Cyclophosphamide (TBI/Cy) and oral busulfan plus cyclophosphamide (BU/Cy) are commonly used myeloablative conditioning regimens in ALL. TBI-based conditioning regimen is considered the standard of care.Aim: To compare the outcomes of allo HSCT using TBI/Cy and BU/Cy conditioning regimens in adult standard and high risk ALL patients in ≥CR1.Material and methods: We retrospectively analyzed the outcome of TBI/Cy and BU/Cy myeloablative conditioning regimen (MAC). BU/Cy cohort included 33 patients (9 females & 24 males) with a median age of 28 years (19-42). Myeloablative BU oral dose was 16mg/kg/4 days & Cy dose was 120 mg/kg IV over 4 days. TBI/Cy cohort included 45 patients (14 females & 31 males) with a median age of 24 years (19-46). TBI dose was 12 Gy fractionated for 5 days & Cy dose was 120mg/kg IV over 4 days. Both cohorts underwent allo-PBSCT at Nasser Institute Hospital, Cairo, between 1997-2010 (TBI group) and 2010-2017 (BU group) (Table1). All patients received graft-versus-host disease (GVHD) prophylaxis using cyclosporine-A (3mg/kg/day IV) from day -1 and methotrexate (15 mg/m2 IV) on day + 1 then 10 mg/m2 IV on days +3, +6 and +11. Post-transplant neutrophil and platelet engraftment were defined by three successive days with absolute neutrophilic count >=1x 109/L and platelet count >=20 x 109/L (without transfusion).Results:In the TBI arm, median time to neutrophil and platelet engraftment was 15 days (9-22) and 15 days (7-47), respectively vs. 14 days (10-22) and 12 days (10-21), respectively in the BU arm. 11/45 TBI patients (24.4 %) developed grade ≥2 acute-GVHD vs. 7/33 (21.2%) patients in the BU arm. Extensive cGVHD was reported in 8/45 (17.7 %) TBI arm vs. 6/33 (18%) patients in the BU arm. Limited cGVHD occurred in 5/45 (11.1%) TBI patients vs. 3/33 (9%) in the BU arm. Cumulative survival was 42.2 % and 15 months OS was 36.7% in the TBI arm vs. 63.6 % and 51.3 % in the BU arm (p= 0.02) (Figure 1). The incidence of sepsis and aGVHD as a cause of transplant related mortality (TRM) were significantly higher in the TBI arm vs. BU arm (p<0.001) (Table 2).Conclusions: Preliminary data demonstrates thatoutcome of BU/Cy is comparable to TBI/Cy as MAC regimen for Allo SCT inadult ALL. With limitation of resources, standard and high risk ALL patients may benefit from allo PBSCT using irradiation-free conditioning regimen associated with lower incidence of sepsis and aGVHD as a cause of transplant related mortality. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Modifications to the “Classical” Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis: Efficacy and Safety Study of a Less Toxic Approach Which Improves the Neurological Condition. a Mexican Perspective

Background: In an effort to reset the immune system, patients with multiple sclerosis (MS) have been autografted with stem cells; we have shown that these grafts can be performed on an outpatient basis with non-frozen peripheral blood hematopoietic stem cells (PBSC) and a conditioning regimen of cyclophosphamide (Cy) and rituximab, the so-called “Mexican method”.Material and methods: Since 2015, all consecutive patients with MS were autografted in two centers in Mexico, following the “Mexican method” (ClinicalTrials.gov identifier NCT02674217), on an outpatient basis and employing PBSC. Mobilization was accomplished with Cy and filgrastim (G-CSF); cumulative dose of Cy was 200 mg/kg. Cy doses were delivered in two separate blocks nine days apart, with the initial aim of mobilizing PBSC and purging lymphoid cells, while the second further purged lymphoid cells and conditioned the graft. After granulocyte recovery, patients were administered rituximab (375 mg/m2) and again (100 mg) every two months, over a 12-mo. period. The extended disability status scale (EDSS) score was assessed every 3 mos. after transplant.Results: 392 patients were autografted; median age was 47 years; time to neutrophil recovery > 0.5 x109/L was 7 days (0-12). The outpatient procedure was completed in 382 patients (97.4%); 10 patients were admitted to the hospital due to neutropenic fever (2), persistent nausea / vomiting (2), iatrogenic pneumothorax (4) and dehydration (2). Transplant related mortality was zero. The 12-mo. progression-free survival was 75%. Improvement or stabilization of the EDSS score at 12 mos. was observed in 75, 72 and 60% of patients with relapsing remitting (RR), secondary progressive (SP) or primary progressive (PP) forms of MS. In patients with a response, EDSS values decreased significantly from 4.83 ± 0.21 to 3.49 ± 0.24 three mo. after grafting (p = 0.007) and from 4.39 ± 0.49 to 3.32 ± 0.50 twelve mo. after grafting (p = 0.05).Conclusions: The Mexican autograft method in MS carries a very low morbidity and no mortality, induces neurological responses in MS, even in variants in which other autograft protocols have not proven useful, such as SP-MS and PP-MS. Additional information and follow-up is needed to further support these observations. DisclosuresGomez-Almaguer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Inhibition of Carbonic Anhydrase-IX As a Novel Strategy to Overcome Hypoxia Mediated Drug Resistance in FLT3/ITD Mutated Acute Myeloid Leukemia

Background: FLT3/ITD mutated (FLT3/ITD+) acute myeloid leukemia (AML) is an aggressive form of blood cancer that carries a poor prognosis due to short remission durations and high relapse rates. Several studies suggest that this poor outcome is due to a small fraction of leukemic cells (LCs) that elude treatment and survive in hypoxic niches of the bone marrow (BM) where they lead to relapse. The identification of molecules capable to target LCs under hypoxic conditions is therefore of utmost importance on the way to find a cure for AML. Our study addresses the effects of pharmacological inhibition of carbonic anhydrase-IX (CA9), a transmembrane protein which functions to maintain a neutral intracellular pH - a key survival mechanism for tumor cells to escape cell death in the hypoxic environment - on AML cells. Targeting CA9 is of particular interest as its expression is confined to only a few normal tissues. CA9 inhibition may therefore show relatively few side effects compared to standard chemotherapies. Methods: Molm14 (M14), M13, OCI-AML3, THP-1 and primary cells (PC) from FLT3/ITD+ AML patients were incubated under normoxic (21% O2) and hypoxic (1% O2) conditions in the presence or absence of Quizartinib (Quiz), Cytarabine (Cy) or the CA9 inhibitors FC531 (FC) or SLC-0111 (SLC). After 48h proliferation and apoptosis assays were performed using MTT assays, annexin V/PI staining and FACS analysis. Cells were also assessed for CA9 and CA12 mRNA expression as well as intracellular pH levels via RT-PCR and fluorescent imaging, respectively. FLT3/ITD+ AML xenografts were generated by injecting M14 cells in the tail vein of NSG mice. Tissue samples were fixed in 10% NBF, embedded in paraffin and stained with H&E and CA9 for pathologic evaluation. Results: CA9 mRNA was upregulated in M14 cells (3.8±1.1-fold; n=3, p<.05) and FLT3/ITD+ AML PCs (215.4±142.7-fold, n=7, p=NS) under 1% compared to 21% O2. Consistently, immunohistochemical staining of M14 xenograft tissues showed multifocal CA9 staining of LCs in the BM and spleen. In M14 cells, inhibition of CA9 with FC conferred substantial, dose-dependent growth inhibitory effects under 1% O2 (IC50=4.04µM) and was significantly more effective than clinically achievable doses of Quiz or Cy in this regard. Under 1% O2, flow cytometry revealed significant induction of apoptosis in FC, but not in Quiz or Cy treated cells (p<.01 vs Cy, p<.01 vs Quiz; n=3). Similar results were obtained in M13 cells. Treatment of M14 cells with 5µM FC under 1% O2 resulted in substantial intracellular pH acidification (ΔpH=-2.7±0.6, p<.05 vs 1% O2, n=3) within 48h. FC synergistically inhibited M14 cell growth when combined with Cy (CI=0.78). To investigate the effects of CA9 inhibition against non-FLT3 mutated cells we incubated OCI-AML3 and THP-1 cells to increasing doses of FC (1-10µM) under 1% O2. We observed that both cell lines were less sensitive to FC (IC50>10µM) compared to M14 and M13 cells. Interestingly, these cell lines displayed higher levels of CA12 mRNA (CA9/12 ratio=0.68 [OCI-AML3] and 0.54 [THP-1]) compared to the FC sensitive cell lines M14 and M13 (CA-9/12 ratio=2.5 and 4.6, respectively). Next, we assessed the effects Cy (1µM), Quiz (100nM) and FC (200µM) against FLT/ITD+ PCs. As expected, Cy and Quiz did not confer cytotoxic effects under 1% O2. Surprisingly, FC was only mildly effective against FLT/ITD+ PCs, which harbored low CA9/12 ratios (<0.5). In contrast, treatment with equimolar doses of SLC, a different class CA9 inhibitor, exhibited substantial anti-leukemic activity under 1% O2 conditions (78.8±1.9% growth inhibition, n=3, p<.05 vs FC, p<.001 vs Cy or Quiz). Conclusions: 1. CA9 is upregulated in FLT3/ITD+ cell lines and PCs under hypoxic conditions. 2. CA9 is expressed in LCs residing in the BM and spleen of FLT/ITD+ AML xenografts. 3. The CA9 inhibitor FC531 confers marked anti-leukemic activity against FLT3/ITD+ AML cells under hypoxic conditions via intracellular pH acidification and acts synergistically with cytarabine. 4. Upregulation of other carbonic anhydrase isoforms, such as CA12, might represent a resistance mechanism against FC531 that can be overcome by different class CA9 inhibitors. 5. Our data suggests that CA9 inhibition has the potential to specifically target LCs residing in hypoxic niches and may be of value as an adjunct to chemotherapy for FLT3/ITD+AML treatment. 6. Our ongoing work evaluates the effects of CA9 inhibition in AML xenografts. DisclosuresNo relevant conflicts of interest to declare.

Patient Data Supports the Rationale of Low Dose Cyclophosphamide to Potentiate the Anti-Myeloma Activity of Daratumumab through Augmentation of Macrophage-Induced ADCP

Introduction:Daratumumab (Dara) is the first CD38-directed monoclonal antibody approved for the treatment of Multiple Myeloma (MM). Dara is thought to have pleiotropic mechanisms of activity, including antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). We focused our attention on potential mechanisms of enhancing macrophage (Mφ)-mediated ADCP and have highlighted that exposure of MM cells to low doses of Cyclophosphamide (Cy) led to a secretory response, which greatly augments Mφ-induced ADCP of Dara-coated MM cells1. Here, we sought to confirm this phenomenon in MM and establish the in-vivo relevance of these findings as seen in newly diagnosed MM patients enrolled in CyBorD-DARA phase 1b clinical trial (NCT02951819).Materials and Methods:Bone Marrow (BM) and peripheral blood (PB) samples were collected in EDTA vacutainers from newly diagnosed MM patients recruited to the CyBorD-DARA clinical trial both prior to- and 24hrs after Cy (150-300mg/m2) treatment. Total lymphocyte, monocyte and neutrophil counts were quantified by flow cytometry. Mononuclear cells were prepared and analysed by 8-colour flow cytometry to quantify monocyte, lymphocyte and plasma subsets as well as determine changes in surface expression profiles following treatment. As previously described, MM cells were conditioned with low-dose Cy for 24hrs. Media containing chemotherapy agents were removed and replaced with fresh media for 24hrs (TCM)1. THP-1 Mφ were conditioned with TCM for up to 48hrs and either characterised or incubated at 2:1 effector target ratio with CFSE labelled MM1S cells ± Cytochalasin D for 18hrs in the presence or absence of Dara/Isotype control. The % of Dara-specific tumour cell clearance was calculated to determine if the mechanism of tumour cell clearance was mediated by Mφ-mediated ADCP.Results:In patients receiving Cy for 24hrs, total BM and PB neutrophil numbers were increased (P<0.01, n=11), lymphocyte numbers were decreased (P<0.01, n=11) while monocyte numbers remain unchanged (P>0.05, n=11). More specifically, an increase in CD19+ B cells (P<0.05, n=6) and a decrease in natural killer cells (NK) (P<0.05, n=6) were observed. Interestingly, the number of Mφ precursor monocytes did not change following Cy treatment in both BM and PB of these patients. As innate immune cells are essential in immune surveillance, coupled with our finding that NK cells are reduced, this highlights a potential role for Mφ in Cy induced anti-tumour activity. Additionally, data previously presented highlighted that exposure of Mφ to TCM from Cy treated MM cells significantly enhanced % of Dara-specific clearance of MM cells) p<0.01). We also confirmed ADCP and ADCC as a likely mechanisms of tumour cell clearance in vitro.Furthermore, we observed a marked reduction in expression of the “don't eat me” antigen, CD47, on chemotherapy-treated MM cells in vitro, which we have now confirmed in MM cells isolated from PB and BM of patients (n=2) following Cy treatment, which may enhance phagocytosis. We also observed increased expression of other targetable markers including BCMA (p<0.05), SLAMF7 (p=0.053), PD-L1 (p<0.001) and PD-L2 (p<0.001) in both RPMI8226 or MM1S MM cell lines following low dose Cy treatment in vitro. To date, we can confirm similar findings for both BCMA (p<0.05, n=5) and SLAMF7 (p<0.05, n=3) in patient samples. Furthermore, our in vitro observation of induced CD64 Fc gamma receptor expression on conditioned Mφ following single-agent Cy treatment has been confirmed in Mφ isolated from both BM (P<0.05, n=11) and PB (P<0.01, n=11) of newly diagnosed MM patients following Cy treatment. This finding emphasizes the potential physiological relevance of our in vitro data, implicating Cy in the potentiation of Mφ mediated ADCP in Dara specific anti-tumour immunity.Conclusions:Observations in Mφ precursors isolated from patients' BM and PB following 24hrs Cy treatment, confirm our previous in vitro findings which suggest a role for Cy potentiation of Mφ mediated ADCP in anti-tumour efficacy of Dara. These findings provide a rationale for the efficacy observed to date when combining Cy with Dara in the ongoing CyBorD-DARA clinical trial. Additionally, increases in BCMA and SLAMF7 highlight the potential of other combination immune therapies to further enhance therapeutic efficacy in these patients.1. Rigalou et al, Blood 2016 128:2101 [Display omitted] DisclosuresChiu:Janssen: Employment. Sasser:Janssen Pharmaceuticals: Employment. Ryan:Janssen: Research Funding. O'Dwyer:Onkimmune Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; GlycoMimetics Inc: Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

Hemorrhagic Cystitis in Patients Undergoing Allogeneic Hematopoietic Cell Transplant with Post Transplant Cyclophosphamide As GvHD Prophylaxis

Hemorrhagic Cystitis in Patients Undergoing Allogeneic Hematopoietic Cell Transplant with Post Transplant Cyclophosphamide As GvHD Prophylaxis

Higher efficacy of intermediate dose cytarabine + G-CSF compared to cyclophosphamide + G-CSF in hematopoietic stem cell mobilization in patients with multiple myeloma.

There are several regimens used in hematopoietic stem cell (HSC) mobilization in multiple myeloma (MM). Cyclophosphamide (Cy) is one of the most commonly used agents, although it does not always result in collecting adequate number of CD34+ cells. Recently, cytarabine (Ara-C) has been proposed as potentially efficient and safe option. Since the data regarding Ara-C in HSC mobilization is limited, the aim of our study was to compare retrospectively the efficiency and toxicity of G-CSF combined with either Ara-C or Cy in MM patients. Of a total of 89 patients, 43 received low or intermediate doses of Cy, and 46 were treated with 800 mg/m2 /day of Ara-C administered for two days. The mean peak of CD34+ cells/ul in peripheral blood was 132 (range, 84-202) in Ara-C and 51 (range, 29-69) in Cy cohort (p < 0.001). The median number of collected CD34+ cells (×106/kg) was 10.3 (range, 4.2-17.9) vs 4.5 (range, 2.7-8.9), respectively (p < 0.001). Mobilization failure was observed in one patient in Ara-C cohort (2%) and in 8 patients treated with Cy (19%) (p = 0.013). In the Ara-C group 98% of patients obtained more than 4×106 CD34+ cells/kg required for tandem transplantation. Moreover, we observed a trend toward increased paraprotein levels measured at transplant compared to before HSC mobilization in Ara-C cohort and significantly higher transfusion rates in that group. Our findings confirm higher HSC mobilization efficacy of Ara-C compared to Cy in MM patients. However, lower transfusions rate and better disease control of Cy may justify its use in some cases.