G-CSF Priming Research Articles

Modelling post-chemotherapy stem cell dynamics in the bone marrow niche of AML patients

Acute myeloid leukemia (AML) is a stem cell-driven malignancy of the blood forming (hematopoietic) system. Despite of high dose chemotherapy with toxic side effects, many patients eventually relapse. The “7+3 regimen”, which consists of 7 days of cytarabine in combination with daunorubicin during the first 3 days, is a widely used therapy protocol. Since peripheral blood cells are easily accessible to longitudinal sampling, significant research efforts have been undertaken to characterize and reduce adverse effects on circulating blood cells. However, much less is known about the impact of the 7+3 regimen on human hematopoietic stem cells and their physiological micro-environments, the so-called stem cell niches. One reason for this is the technical inability to observe human stem cells in vivo and the discomfort related to bone marrow biopsies. To better understand the treatment effects on human stem cells, we consider a mechanistic mathematical model of the stem cell niche before, during and after chemotherapy. The model accounts for different maturation stages of leukemic and hematopoietic cells and considers key processes such as cell proliferation, self-renewal, differentiation and therapy-induced cell death. In the model, hematopoietic (HSCs) and leukemic stem cells (LSCs) compete for a joint niche and respond to both systemic and niche-derived signals. We relate the model to clinical trial data from literature which longitudinally quantifies the counts of hematopoietic stem like (CD34+CD38-ALDH+) cells at diagnosis and after therapy. The proposed model can capture the clinically observed interindividual heterogeneity and reproduce the non-monotonous dynamics of the hematopoietic stem like cells observed in relapsing patients. Our model allows to simulate different scenarios proposed in literature such as therapy-related impairment of the stem cell niche or niche-mediated resistance. Model simulations suggest that during the post-therapy phase a more than 10-fold increase of hematopoietic stem-like cell proliferation rates is required to recapitulate the measured cell dynamics in patients achieving complete remission. We fit the model to data of 7 individual patients and simulate variations of the treatment protocol. These simulations are in line with the clinical finding that G-CSF priming can improve the treatment outcome. Furthermore, our model suggests that a decline of HSC counts during remission might serve as an indication for salvage therapy in patients lacking MRD (minimal residual disease) markers.

Venetoclax Plus Azacitidine, Low Dose Cytarabine, Aclarubicin and G-CSF (VA-CAG Regimen) for Newly Diagnosed Young Patients with Acute Myeloid Leukemia: A Prospective, Multicenter, Single-Arm Phase 2 Clinical Trial

Background: Venetoclax, in combination with hypomethylating agents or low-dose cytarabine, is approved for newly diagnosed acute myeloid leukemia (AML) patients who are over 75 years of age or not eligible for intensive chemotherapy, while the efficacy of venetoclax-based regimens in young fit AML patients still needs to be further explored. Although the combination of anthracycline and cytarabine has achieved a remission rate of about 70% in these patients, the low complete response rate in adverse-risk patients, strong adverse reactions, and long duration of myelosuppression limited the clinical benefit. We presumed that venetoclax plus azacitidine in combination with low-intensity regimens CAG (G-CSF priming, low dose cytarabine, and aclarubicin) could have a better composite complete remission with an acceptable safety profile. Methods: Patients who were newly diagnosed with AML aged from 18 to 65 years without active infection, bleeding and severe cardiopulmonary insufficiency were enrolled. Participants received induction as azacitidine 75 mg/m 2 subcutaneously once daily on days 1-7, cytarabine 10mg/m 2 subcutaneously q12h on days 1-7, aclacinomycin 12 mg/m 2 on days 1,3,5,7, granulocyte colony-stimulating factor 5µg/kg on days 1-7, and venetoclax orally once daily (100 mg d1, 200 mg d2, 400 mg d3-21). Venetoclax dose was adjusted based on concomitant CYP3A inhibitors. The efficacy were evaluated by bone marrow morphology and flow cytometry on day 28 cycle 1. The primary endpoints were safety and the composite complete remission rate. This study was approved for ethical review and registered with ClinicalTrials.gov (NCT05662956). Results: Twenty-six patients from eight sites were enrolled between January 1, 2023, and June 20, 2023. The median age was 52 years (range, 18-63years). Thirteen patients (50.0%) had adverse risk, 7 patients (26.9%) had intermediate risk, and 6 patients (23.1%) had favorable risk. All 26 patients (100%) achieved complete remission after cycle 1, and 21 patients (80.8%) was MRD negative by flow cytometry. Two patients (7.7%) were treated with venetoclax for 14 days due to ineffective platelet transfusion and severe infection during treatment. Twenty-two patients (84%) used azole drugs to prevent fungal infection. The median myelosuppression time was 13 days (range, 5-22days), and the median red blood cell and platelet transfusions were 5.5u (range, 0-11.5u) and 30u (range, 0-170u), respectively. Eleven patients (42.3%) experienced infection (including pulmonary fungal infection, oral cold herpes, novel coronavirus infection, etc.), and two of them (7.7%) had a breakthrough invasive fungal infection, but with good management. The most common grade 3/4 adverse events included bone marrow suppression (92.3%), liver injury (19.2%), gastrointestinal reaction (11.5%) and coagulation dysfunction (3.8%).No treatment-related death occurred. Conclusion: VA-CAG regimen achieved high rates of complete remission and MRD negative in young adult fit patients with newly diagnosed AML. This combined regimen has an acceptable safety profile, with low incidence of infection and transfusion dependence.

Granulocyte colony-stimulating factor priming improves embryos and pregnancy rate in patients with poor ovarian reserve: a randomized controlled trial

BackgroundGranulocyte colony-stimulating factor (G-CSF) administration increased ovarian preantral follicles and anti-Müllerian hormone (AMH) in animal models with diminished ovarian reserve. We investigated whether G-CSF priming before treatment with assisted reproductive technology (ART) improved embryo development and pregnancy rate while increasing serum AMH in patients with poor ovarian reserve.MethodsIn this prospective randomized open-label controlled trial, 100 patients 20 to 42 years old with AMH below 2 ng/mL were randomized to priming or control groups (50 patients each). None had over 1 ART failure, day-3 follicle-stimulating hormone (FSH) above 30 IU/L, uterine anomalies, or a partner with azoospermia. All patients initially underwent conventional infertility treatment for 2 consecutive cycles in which the priming group but not controls received a subcutaneous G-CSF priming injection during the early luteal phase. Each group then underwent 1 cycle of in vitro fertilization/intracytoplasmic sperm injection and fresh embryo transfer (IVF/ICSI-fresh ET), followed by cryopreserved ET if needed until live birth or embryo depletion. AMH was measured before and after priming.ResultsFertilization rate, embryonic development, and implantation rate by fresh ET were significantly improved by priming. Clinical and ongoing pregnancy rates by IVF/ICSI-fresh ET were significantly higher with priming (30% and 26% in 47 ART patients; 3 delivered with conventional treatment) than in controls (12% and 10% in 49 ART patients; 1 dropped out). With priming, significantly more patients achieved cryopreservation of redundant blastocysts. The cumulative live birth rate was 32% in 50 patients with priming, significantly higher than 14% in 49 controls (relative risk, 2.8; 95% confidence interval, 1.04–7.7). Infants derived from priming had no congenital anomalies, while infant weights, birth weeks, and Apgar scores were similar between groups. Among 4 variables (age, day-3 FSH, AMH, and priming), logistic regression significantly associated age and priming with cumulative live birth. Priming significantly increased serum AMH. No adverse effects of priming were observed.ConclusionG-CSF priming improved embryonic development and pregnancy rate during ART treatment and increased AMH in patients with poor ovarian reserve. Enhanced preantral follicle growth likely was responsible.Trial registrationUMIN registration in Japan (UMIN000013956) on May 14, 2014. https://www.umin.ac.jp/ctr/index.htm.

Homoharringtonine Synergizes Strongly with Venetoclax Against the Early T-Cell Progenitor Acute Lymphoblastic Leukemia：from Bench to Bed

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with poor outcome and novel therapies are urgently needed. Venetoclax (VEN), a BCL2 inhibitor, has been approved by FDA for AML. Combination of VEN and demethylating drugs or targeted therapies such as MCL1 inhibitor showed promising effect in AML. Loucy, an ETP-ALL cell line, was also reported high expression of BCL2. But little is known about VEN combination treatment in ETP-ALL. Our data showed that homoharringtonine (HHT), a plant alkaloid, downregulated MCL1 in T-ALL. Herein, we examined the treatment effect of VEN combined with HHT on ETP-ALL using murine model and patient samples. First, we compared BCL2 levels and treatment effect of VEN alone on ETP-ALL (i.e., Loucy) and T-ALL (i.e., Jurkat, Molt4, and CCRF-CEM) cell lines. We observed higher levels of BCL2 and lower IC50 values (IC50 at 24h: 0.093 uM vs.>1uM) in Loucy vs T-ALL cells. Similar results were found in human samples, with IC50 of 0.539uM in ETP-ALL sample vs 3.165-23.25uM in T-ALL samples treated with VEN for 24h. Next, to determine whether the combination of VEN and HHT shows a synergistic effect on ETP-ALL, Loucy cells were treated with a serial concentration of VEN (range: 0.025-0.1uM) and HHT (range:10-40ng/mL) alone and in combination for 24h. VEN and HHT combination showed significant synergism in reducing cell viability, with a mean combination index (CI) of < 0.1. We observed similar results in patient samples, with mean CI lower than 0.01 indicating highly synergistic effect. To assess this in vivo, a cohort of ETP-ALL mice generated by transplanting Loucy cells into NSG mice were randomly divided into 4 groups and treated with vehicle, HHT (1 mg/kg, ip), VEN (25 mg/kg, oral gavage) or HHT+VEN combination. HHT and VEN combination significantly prolonged survival of the ETP-ALL mice compared to either drug alone or vehicle (median survival of combination vs VEN vs HHT vs control: 113.5 vs 95.5 vs 85.5 vs 69.0 days, p<0.01), suggesting a synergistic effect of HHT and VEN in vivo. To identify the mechanical basis for the synergy of VEN and HHT in ETP-ALL, we performed single-cell RNA-seq using primary patient ETP-ALL cells treated with vehicle, HHT, VEN or their combination for 24h. Based on the gene expression profile, we identified a cluster of ETP-ALL cells sensitive to HHT , and a cluster only sensitive to combination of VEN and HHT which showed higher levels of Bcl2 and Mcl1 and upregulated anti-apoptosis signal by gene set enrichment analysis (GSEA), suggesting intra-tumoral heterogeneity of ETP-ALL blasts. These results suggest that HHT or VEN alone is not sufficient to induce apoptosis in the resistant cells and combination of HHT and VEN could induce apoptosis by reducing MCL1 expression in these cells which was confirmed by western blot. Further, with the promising results from bench work and given that both HHT and VEN are approved anti-leukemia drugs, we translated these findings into clinic. The HAG (HHT, low dose Cytarabine, G-CSF priming) regimen has been widely used in China for the treatment of AML for 15 years and proved to be effective and safer than intensive chemotherapy. Based on above findings, with informed consent from patients, we applied venetoclax plus HAG(V-HAG) regimen to 3 relapsed/refractory(R/R) ETP-ALL patients and 4 newly diagnosed (ND) patients, including an 82-year-old refractory patient. CR/CRi rates after the first cycle treatment were 100%, which was significantly higher than the current induction therapy (CR rate ranging from 33.3-55.9% after the first induction cycle). Except for the 82-year-old patient who refused post-remission treatment, all the remaining 6 patients remained in remission until today. Of note, no clinical manifestations of tumor lysis syndrome were observed. The median time to absolute neutrophil count recovery of >500/μL and >1000/μL was 13 (range: 0-22) and 16 (range: 0-24) days, and to platelet count recovery of > 20,000/ μL was 16 (range: 0-21) days, indicating this regimen was relatively safe and well tolerated. In conclusion, our findings demonstrate that HHT and VEN showed synergistic effect on ETP-ALL and HHT/VEN based therapy is a promising and safe regimen in newly diagnosed and R/R ETP-ALL patients. With these promising results from bench to bedside, 2 multi-center phase Ib/II clinical trials using V-HAG in patients with R/R ETP-ALL and ND-ETP have been initiated at our institution.

The Effects of Decitabine Combined with All-Trans Retinoic Acid on the Number of Immune Cells in Myeloid Neoplasms

To investigate the effects of decitabine (DEC) combined with all-trans retinoic acid (ATRA) on the number of immune cells, efficacy and adverse reactions in the treatment of myeloid neoplasms patients. Eighty-four patients with myeloid tumors, including AML, MDS-EB-1 or MDS-EB-2 treated by the regimen containing decitabine in our hospital from January 2009 to October 2019 were enrolled and retrospectively analyzed, among the patients, 21 patients treated with DEC alone, 24 patients treated with DEC combined with ATRA (DEC/ATRA) and 39 patients treated with DEC combined with G-CSF priming regimen (DEC/priming). The changes of peripheral blood immune cell levels before and after treatment of the patients between the three groups were compared, and the differences in clinical efficacy and adverse reactions of the patients between the three groups were also compared. There was no statistical differences in the number of immune cells among the patients in the three groups before treatment (P>0.05). NK cell levels decreased significantly in the patients in DEC and DEC/ATRA group after treatment (P<0.05); After treatment, the levels of CD8+ and CD3+T cells in the patients treated by DEC /priming regimen significantly increased (P<0.05), while the levels of CD3-HLA-DR+ B cells significantly decreased (P<0.05). The overall response rate (ORR) of the patients in DEC/ATRA group (75%) and DEC/priming group (74.36%) was significantly higher than 42.86% in DEC monotherapy group, and the differences showed statistically significant (P<0.05), while the ORR between the patients in DEC/ATRA and DEC/priming group showed no statistic differences (P>0.05). There were no statistical differences in overall survival (OS) and incidence of bleeding between the patients in the three groups (P>0.05). The incidences of grade 3 to 4 bone marrow suppression and the infection rate of the patients in DEC monotherapy and DEC/ATRA group were significantly lower than that in DEC/priming regimen group after treatment (all P<0.05), however, there was no statistical difference between DEC monotherapy and the DEC/ATRA group. The efficacy of DEC/ATRA on myeloid neoplasms is comparable to that of DEC/priming regimen, and the anti-myeloid tumor effect of DEC/ATRA regimen may be related to the regulation of NK cells and T cells.

The Synergy of Vitamin C with Decitabine Activates TET2 in Leukemic Cells and Significantly Improves Overall Survival in Elderly Patients with Acute Myeloid Leukemia

Background: Decitabine is widely used in the treatment of acute myeloid leukemia (AML) in elderly patients. Low-dose ascorbic acid (Vitamin C) has also been indicated to induce DNA demethylation at the cellular level. Our previous study demonstrated its combined effect and safety with low-dose decitabine prior to aclarubicin andcytarabine (DCAG) in elderly patients with AML, in which it appeared to achieve a complete remission (CR) rate of 76.3% and a median overall survival of 10 months. Pharmacological concentrations of Vitamin C (10-1,000μM) induced a time- and dose-dependent increase in 5-hmC. However, little is known whether low-dose Vitamin C has a synergistic effect with decitabine in clinic. Methods: The effect of combined low-dose Vitamin C and decitabine on cell proliferation, the cell cycle, apoptosis and the expression level and activity of TET2 was investigated in HL-60 and NB-4 human leukemic cells. Additionally, we analyzed the clinical outcomes of 73 elderly AML patients who received A-DCAG (intravenous Vitamin C [IVC] plus DCAG [ n=39]) or DCAG (n=34) treatment. Patients received one of the two induction courses: DCAG or A-DCAG. DCAG consisted of 15mg/m2 of decitabine (intravenously, days 1-5) and 300 μg/day of granulocyte colony-stimulating factor (G-CSF, days 0-9) for priming its combination with 10 mg/m2 of cytarabine (q12h, days 3-9), 8 mg/m2 of aclarubicin (days 3-6, DCAG), or IVC (50-80 mg/kg/day, days 0-9; A-DCAG). The G-CSF priming was discontinued when white blood count (WBC) was ≥20×109/L. Results: We found that low-dose Vitamin C and decitabine has a synergistic efficacy on proliferation, apoptosis and TET2 activity, compared to drug-alone treatment in HL60 and NB4 cell lines in vitro . In clinic, feasibility and safety evaluations revealed that patients who received A-DCAG regimen have a higher complete remission (CR) rate than those who received the DCAG regimen (79.92 % vs. 44.11%; P=0.004) after one cycle of chemotherapy. IVC admission decision appeared to beat the discretion of attending physicians. The median overall survival (OS) was better in the A-DCAG group compared with the DCAG group (15.3 months vs. 9.3 months, P=0.039). Patients with adverse cytogenetics did benefit from CR. There was no clinically significant additional toxicity observed with the addition of IVC. Conclusion: On the basis of these results, the addition of IVC at low doses to DCAG appeared to improve CR and prolong OS, compared with DCAG, in elderly patients with AML. DisclosuresNo relevant conflicts of interest to declare.

Risk-Adapted Intensive Chemotherapy for Primary ACUTE Myeloid Leukemia during the Last 25 YEARS: Increase in Complete Remission RATE, Hematopoietic Cell Transplantation Access and Decrease in Relapse Incidence Have LED to Improved Survival

Risk-Adapted Intensive Chemotherapy for Primary ACUTE Myeloid Leukemia during the Last 25 YEARS: Increase in Complete Remission RATE, Hematopoietic Cell Transplantation Access and Decrease in Relapse Incidence Have LED to Improved Survival

Peripheral Blood to Marrow Collection White Cell Count Ratio As an Indicator of Harvest Quality and Transplant Outcomes in G-CSF-Primed Marrow Grafts

Background: G-CSF-primed bone marrow (G-BM) is used in some centers as the product of choice in related donor transplants because of lower rates of GVHD and fast engraftment (Morton 2001; Deotare et al. 2015). Marrow collections substantially diluted with peripheral blood may, however, compromise the GVHD-sparing effect of G-BM. Concerns over the variability in the quality of bone marrow harvest have been reported (Spitzer et al. 1994; Prokopishyn et al. 2019). We explored the role of a simple novel quality indicator, i.e peripheral blood to marrow harvest WBC ratio, as a predictor of GVHD risk. Materials and methods: We analysed 156 successive bone marrow harvests for patients suffering from severe thalassemia using G-BM donated by fully matched (10/10 or 12/12) related donors (143 siblings and 13 mothers) at Sankalp-People Tree Centre for Paediatric BMT - Bangalore (80 patients) and Sankalp-CIMS Centre for Paediatric BMT - Ahmedabad (76 patients). G-CSF priming was done for 5 days before the transplant with 10 mcg/kg/day in 2 divided subcutaneous doses and peripheral counts were obtained within 24 hours of collection. G-BM was collected in bags with 20 ml 50 U/ml heparin for the first 200 ml and further 10ml/100ml of marrow. We defined quality of marrow as follows: Quality of Marrow = WBC count of G-CSF primed BM / Peripheral WBC count of donor Quality of Marrow &lt; 0.9 was categorised as poor as it signifies that the concentration of WBCs in bone marrow is same or lesser than the pre-harvest peripheral counts. EBMT criteria was used for grading GVHD. We calculated the odds of aGVHD based upon the quality of marrow. Beside marrow quality, the sex of the recipient seemed to impact GVHD rates and thus regression analysis was used to study the impact of G-BM quality independent of other factors. Statistics were performed on R software (version 3.5.x). Mann Whitney and Fisher's exact tests were used to compare numeric and categorical data respectively. Results: Of the 156 harvests, 18 had poor marrow quality; 33% (6 of 18) of these developed acute GVHD grade II-IV as opposed to 9% (13 of 138) (see Figure 1). Between those who developed GVHD and those who did not, marrow quality (p=0.012), patient sex (p=0.006) and sex mismatch status (p=0.014) were significantly different. Differences in total cell dose (p=0.65), transplant centre (p=0.62), bone marrow cell count (p=0.14) and peripheral cell count before harvest (p=0.72), donor age at BMT (p=0.42), relationship with the donor (p=1.0) and average volume of marrow per draw of syringe (p=0.23) showed no statistical significance. Variables with significant difference (sex mismatch and bone marrow quality) were entered in the Cox's proportional hazards model for acute GVHD II-IV which showed that poor marrow quality is the most significant independent risk associated with a hazard ratio of 3.6 (p=0.009), followed by male to male transplants which were associated with a hazard ratio of 10.6 (p=0.03) See Table 1. Further, poor quality marrow was associated with lower cell count in marrow (p&lt;0.001), higher peripheral counts (p&lt;0.001), higher collection volume (p=0.035) and was seen more in one centre compared to the other (p&lt;0.001). Donor age (p=0.81), donor's relationship (p=1.0), donor sex (0.62), donor's carrier status (p=0.64), donor weight (p=0.85) and average volume of marrow per draw of syringe (p= 0.76) were not statistically significant. Discussion: The role of tolerance induced by mesenchymal stem cells in reduction (Aggarwal 2005) and even the treatment of GVHD (Le Blanc et al. 2004; Baron and Storb 2012) has been reported elsewhere and supports our findings though this is yet to be established (Fisher et al. 2019). Simple quality control of BM could be highly beneficial in all settings in minimising acute GVHD. This is particularly important in low- and middle-income countries where GVHD can be very challenging (Saleem et al. 2018) and is associated with high costs of management and prolonged hospitalisation (Grubb et al. 2016). Our indicator is accurately measurable, inexpensive, precise and timely; further it does not involve any sophisticated equipment (flow cytometry for CD counts), thus may be widely applicable and usable. Conclusion: Quality of BM harvest, as indicated by PB to BM WBC ratio, is a simple, effective and inexpensive informative indicator in the context of G-BM. Poorer quality of marrow collection may be contributing to increased risk of acute GVHD. Disclosures No relevant conflicts of interest to declare.

Comparable Leukemic Cells Reduction but Improved Normal Hematopoietic Regeneration with G-CSF Priming Plus Low Dose Chemotherapy Regimen Relative to Standard Chemotherapy in AML Mice Model

Pediatric acute myeloid leukemia (pAML) is a heterogeneous disease. The clinical outcome of pAML has improved significantly over the past four decades with current long-term survival rates of ~70%. This improvement is due to intensification of chemotherapeutic regimens, better risk-group stratifications, better salvage at relapse, and improved supportive care. Nevertheless, novel therapies are still needed as the cure rates for some subtypes of pAML remain unacceptably low. In addition, the profound marrow suppression caused by near-myeloablative induction therapy may lead to life-threatening complications, such as severe infections, which not only prolong the hospitalization but also cause significant morbidity and mortality during or after treatment. Moreover, intensive chemotherapy usually affects the patient's quality of life and they can experience low physical, emotional, or social function. To determine the impact of reducing the intensity of induction chemotherapy on the outcome of pAML, we recently performed a study using G-CSF priming plus low-dose chemotherapy (GLDC) to treat pAML, compared with standard chemotherapy (SDC) during induction. Our study showed that complete remission (CR), 4-year event free survival (EFS), overall survival (OS), and cumulative incidence of relapse (CIR) rates of 46 pAML patients, initially given a GLDC for the first induction remission, were comparable to those of 94 children admitted to the same hospital and given SDC. However, GLDC showed a much lower toxicity than SDC. After the first course of induction, the recovery of white blood cell (WBC) and platelet counts were significantly faster in patients receiving GLDC than SDC. Grade III or IV infectious complications occurred in 4 (8.7%) patients receiving GLDC and 23 (24.4%) patients receiving SDC, and 2 patients receiving SDC died of complications (Hu Y. et al, 2019). Even though the clinical efficacy of GLDC has been seen in multiple clinical trials, the detailed mechanisms remain unclear. Some clinical and pre-clinical studies showed that G-CSF could push AML cells into cell-cycle, accelerate WBC and platelet recovery even when used after induction chemotherapy, and reduce the viability of AML cells when co-cultured with BM stroma, but not cultured alone. However, all of these studies were not established with LDC. To determine the potential mechanism(s) of GLDC in pAML, we established Mll-Af9 transplanted chimeric mice to develop AML, and we assessed the clearance of leukemic cells and the impact on normal cells by GLDC or SDC. Based on previous studies, we used combined cytarabine (100mg/kg) and doxorubicin (3mg/kg) (DA) in mice as SDC, half dose of SDC as LDC, and G-CSF (300ug/kg) plus half dose of SDC as GLDC in our study design. The G-CSF priming times were inconsistent in previous studies. To establish the best priming effect, especially for the stem cells, we first analyzed the cell cycle stimulation by G-CSF to the SLAM hematopoietic stem cells (SLAM-HSCs) and the more quiescent stem-like HSC population (endothelial protein C receptor expressing, EPCR+ HSCs) in WT mice. We found that G-CSF can stimulate SLAM-HSCs and EPCR+ HSCs into cell-cycle as early as 12hrs and 24hrs post G-CSF injection, so we used G-CSF one day priming before the chemotherapy estimating that even the deeper quiescent stem cells have been stimulated into the cell-cycle at that time point. We then arranged the Mll-Af9 transplanted mice with similar disease burden into 5 groups to receive different treatments for 6 days: LDC, GLDC, SDC, and also the G-CSF or the PBS groups as control (G-CSF was given on D1-D6, DA was given on D2-D6). We found that GLDC and SDC had similar clearance to the leukemic cells (CD45.2+), which were better than LDC, as observed 2 days after the treatment. However, the recovery of normal cells (CD45.1+) was significantly faster in the GLDC group compared with LDC and SDC groups. We also found that G-CSF could simulate the differentiation of both leukemic cells and normal cells, shown by the increase of the CD11b+Ly6G+ population of both CD45.2+ and CD45.1+ cells in the G-CSF group compared to the PBS group. Studies to further elucidate the additional mechanisms of GLDC are still ongoing. In conclusion, our clinical and mouse modeling studies consistently suggest that GLDC promotes leukemia cells clearance and normal hematopoietic regeneration simultaneously, which may be a better regimen for pAML. Disclosures No relevant conflicts of interest to declare.

Deficiency of Socs3 leads to brain-targeted EAE via enhanced neutrophil activation and ROS production.

Dysregulation of the JAK/STAT signaling pathway is associated with Multiple Sclerosis (MS) and its mouse model, Experimental Autoimmune Encephalomyelitis (EAE). Suppressors Of Cytokine Signaling (SOCS) negatively regulate the JAK/STAT pathway. We previously reported a severe, brain-targeted, atypical form of EAE in mice lacking Socs3 in myeloid cells (Socs3ΔLysM), which is associated with cerebellar neutrophil infiltration. There is emerging evidence that neutrophils are detrimental in the pathology of MS/EAE, however, their exact function is unclear. Here we demonstrate that neutrophils from the cerebellum of Socs3ΔLysM mice show a hyper-activated phenotype with excessive production of reactive oxygen species (ROS) at the peak of EAE. Neutralization of ROS in vivo delayed the onset and reduced severity of atypical EAE. Mechanistically, Socs3-deficient neutrophils exhibit enhanced STAT3 activation, a hyper-activated phenotype in response to G-CSF, and upon G-CSF priming, increased ROS production. Neutralization of G-CSF in vivo significantly reduced the incidence and severity of the atypical EAE phenotype. Overall, our work elucidates that hypersensitivity of G-CSF/STAT3 signaling in Socs3ΔLysM mice leads to atypical EAE by enhanced neutrophil activation and increased oxidative stress, which may explain the detrimental role of G-CSF in MS patients.

Relationship between white blood cell count elevation and clinical response after G-CSF priming chemotherapy for acute myeloid leukemia.

We retrospectively analyzed the relationship between white blood cell (WBC) count elevation after priming and clinical response in 115 patients with AML (61 untreated and 54 relapsed or refractory) treated with low-dose cytarabine, aclarubicin, and G-CSF priming. Receiver operating characteristic curve analysis showed that the ratio of maximum WBC count to pretreatment WBC count (WBCratio) was most strongly associated with complete remission (CR) in previously untreated patients among several parameters we analyzed in this study; however, the prediction accuracy was not clinically significant considering the area under the curve of 0.694. Based on the cutoff value of the WBCratio, CR rate and event-free survival in the high WBCratio group were significantly better than those in the low WBCratio group in untreated patients. Regarding the WBC differential counts, a high ratio of the maximum to pretreatment value of neutrophils rather than that of peripheral blasts was associated with a superior CR rate. In addition, an increase in blasts after G-CSF priming had a significant negative impact on CR rate in untreated patients. In conclusion, an increase in blast counts after G-CSF priming was not predictive of achieving CR.

Role of G-CSF priming of donor marrow prior to transplant in low risk HLA matched sibling transplant in children with severe thalassemia

Role of G-CSF priming of donor marrow prior to transplant in low risk HLA matched sibling transplant in children with severe thalassemia

Clofarabine-Based Consolidation Improves Relapse-Free Survival of Younger Adults with Non-Favorable Acute Myeloid Leukemia (AML) in First Remission: Results of the Randomized ALFA-0702/Clara Study (NCT 00932412)

Background: Clofarabine has been shown as effective as single-agent or in combination with cytarabine in various populations of AML patients. Nevertheless, two randomized studies failed to demonstrate prolonged survival when compared to low-dose cytarabine in older AML patients (Burnett, 2013) or intermediate-dose cytarabine (IDAC) in patients aged 55y or more with relapsed/refractory AML (Faderl, 2012). As this was likely due to excessive toxicity and poor compliance in these difficult-to-treat populations, we made the hypothesis that a more apparent benefit might be observed during post-remission therapy. We thus randomly assessed the role of CLARA combination (clofarabine + IDAC) versus high-dose cytarabine (HiDAC) as consolidation cycles in younger AML patients.Methods: Between 2009 and 2013, 713 patients aged 18-60y old with de novo AML (CBF-AML excluded) were enrolled in the ALFA-0702 trial by 33 French centers. They received a timed-sequential induction with daunorubicin (60 mg/m2, day 1-3; 35 mg/m2, day 8-9), cytarabine (500 mg/m2 CI, day 1-3; 1 g/m2/12h bolus, day 8-10) and G-CSF priming. A first salvage with idarubicin and HiDAC may be proposed to patients not achieving CR/CRp after this first course. Patients in CR/CRp with non-favorable AML (according to ELN classification) or those who needed the salvage (late CR/CRp) were eligible for: 1) allogeneic stem cell transplantation (SCT) if they had a sibling or fully matched unrelated donor; or 2) randomization for consolidation with three HiDAC (3 g/m2/12h cytarabine, day 1/3/5; G-CSF priming) or CLARA (30 mg/m2 clofarabine, day 2-6; 1 g/m2/12h cytarabine, day 1-5; G-CSF priming) cycles if no identified donor. Primary endpoint was relapse-free survival (RFS), patients receiving allogeneic SCT in first CR/CRp after randomization (late donor identification) being censored at SCT time. Secondary endpoints were cumulative incidence of relapse (CIR) and death in first CR/CRp (CID), overall survival (OS), and safety. Anticipating a 35% SCT rate in randomized patients, 230 patients should be randomized to demonstrate a 20% gain in 2-year RFS. A sensitivity analysis without SCT censoring was planned.Results: Among 468 CR/CRp patients of interest (465/552 non-favorable AML + 3/161 favorable AML with late CR/CRp), 221 were randomized between HiDAC (n=114) and CLARA (n=107) consolidation and 247 were not randomized for the following reasons: 181 had an identified donor for SCT; 19 had favorable ELN genotype even if no evidence of normal karyotype; and 47 were negatively selected (13 early relapses, 1 early death, 18 AEs, 15 drop-out). The ITT analysis was conducted in these 221 eligible patients (median age, 48y; 136 intermediate-risk, 85 unfavorable-risk; 18 late CR/CRp; without imbalances between randomization arms), the remaining 9 randomized patients having non-eligibility criteria (6 favorable-risk AML and 3 not in CR/CRp). Among these patients, the rate of SCT in first CR/CRp was higher than anticipated (50%; 55 patients in each arm). With a median follow-up of 37.4 months and SCT censoring, 2-year RFS was 52.1% (40-68) in the CLARA arm versus 30.5% (20-46) in the HiDAC arm (HR, 0.62 [0.39-0.99]; p= 0.042). This was due to a lower CIR in the CLARA arm (44.0 versus 67.7%; p= 0.023) with similar CID (3.9 versus 1.9%, p= 0.60). At 2 years, OS was 68.1% (56-82) in the CLARA arm versus 49.8% (38-66) in the HiDAC arm (p= 0.18). Interestingly, the gain in CIR and RFS was observed in both intermediate- and unfavorable-risk groups and persisted after adjustment on AML risk and age. As expected, these gains were lower when not censoring at SCT (2-year RFS, 57.8 versus 45.6%; p= 0.12). In the 110 randomized patients allografted in first CR/CRp, no differences in post-SCT outcome were observed according to randomization arm. In Mantel-Byar time-dependent analysis, longer RFS was observed after SCT in the HiDAC arm (HR, 0.45; p= 0.004), while not in the CLARA arm (HR, 0.71; p= 0.26). Finally, CLARA cycles were associated with higher hematologic toxicity, more infections and more liver toxicities than HiDAC cycles, especially after cycle 1.Conclusions: As compared to HiDAC, clofarabine-based consolidation improved RFS in younger patients with intermediate- and unfavorable-risk AML in first CR/CRp. CLARA combination might thus become a new standard for post-remission therapy in these patients, especially in those who may not benefit from allogeneic SCT. DisclosuresOff Label Use: CLOFARABINE is not currently approved for this indication.. De Botton:Agios pharmaceuticals: Research Funding. Vey:Janssen: Honoraria; Roche: Honoraria; Celgene: Honoraria. Dombret:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Clinical outcomes and graft characteristics in pediatric hematopoietic stem cell transplantation: Effect of granulocyte-colony stimulating factor priming

In this study, we aimed to determine the effect(s) of G-CSF priming on graft and transplantation parameters and compare these findings with those obtained without priming.A total of 64 pediatric patients transplanted from HLA-matched family donors were enrolled in the study. Twenty-nine patients received G-CSF primed marrow (G-BM group) and 35 patients received steady state bone marrow (S-BM group). Number of total nucleated cells (TNC) and CD34+ cells, CFU-GM colony number, neutrophil and platelet engraftment times, total length of stay in hospital, overall and disease free survival, and occasions of acute and chronic GvHD has been compared between these two groups.Granulocyte colony stimulating factor primed bone marrow (G-BM) yielded higher numbers of CD34+ cells, TNCs, and CFU-GM colony numbers compared to those obtained in S-BM. The neutrophil engraftment time, platelet engraftment time, length of stay in hospital, overall survival and disease free survival were not different between G-BM and S-BM groups. Also the cumulative incidence of grades II–IV acute and chronic GvHD were similar. It was observed that the use of G-CSF did not increase the risk of acute or chronic GvHD.We concluded that use of G-CSF for stem cell mobilization is an effective and safe method in children.

Improved Outcomes of Haploidentical Blood and Marrow Transplantation with Giac-3 Protocol That Haploidentical Bone Marrow from the Second Donor As the Third Party Cells

Introduction: With GIAC regimen (G: G-CSF priming; I: intensified immune-suppression; A: ATG application; C: combination of peripheral blood stem cell and bone marrow (BM) as graft), haploidentical blood and marrow transplantation (haplo-BMT) has been an important alternative option for patients with hematological malignancies who need urgent transplant but without HLA matched either sibling or unrelated donor (Dao-Pei Lu et al., Blood 2006; 107:3065). In a murine model, we have first demonstrated that the animals transplanted with three mixed cells (A+B+C to A) were survived much longer due to milder acute GVHD (aGVHD) compared with the mice transplanted with single allogeneic BM (B to A). Our previous clinical study has shown that with GIAC-3 protocol (GIAC with the third party cells in order to induce immune-tolerance) that unrelated cord blood (UCB) as the third party cells, the incidences of aGVHD, especially for severe aGVHD, and also treatment-related mortality in haplo-BMT have been significantly reduced.Objective: In present clinical study, we examine whether haploidentical bone marrow (BM) from the second donor that substitutes for UCB as the third party cells could also reduce aGVHD and improve survival in haplo-BMT setting.Patients and Methods: Between April 2012 and June 2013, total 158 haplo-BMT patients with hematological malignancies were enrolled. The median age was 18 (1.8 to 56) years old. The diagnosis included AML (46.2%), ALL (42.4%), lymphoma (3.8%), CML (2.5%), MDS (1.9%), acute mix leukemia (1.9%), JCMML(0.6%)and plasma cell leukemia (0.6%). Transplants at CR1, ≥CR2 or CML-AP, and advanced disease (refractory/relapsed acute leukemia or CML-BC) were 34.8%, 24.7% and 40.5%. All patients received unmanipulated blood and marrow transplant after BUCy2 or CyTBI plus antithymocyte globulin as pre-conditioning. Fludarabine was substituted for cyclophosphamide in some patients due to impaired organ function or high tumor burden. Cyclosporine, short-term Methotrexate, and Mycophenolate mofetil were employed for GVHD prophylaxis. 1ml/kg (recipient’s body weight) haploidentical BM from the second donor was infused right after haplo-BMT as the third party cells.Results: All patients but two achieved engraftment. One case had primary graft failure and the other one had secondary graft failure. Both patients obtained durable hematopoietic reconstitution after the second BMT. Low levels of the third party cells were detected in a few patients at early stage after transplantation. No long-term third party cell engraftment was found. The cumulative incidences of grade II to IV aGVHD and grade III to IV aGVHD were 30.5%, 14.0%, respectively. With the median follow-up time of 17 months, two-year leukemia-free survival (LFS) rates in CR and NR cases were 73.2%, 41%, respectively, and 2-year overall survival (OS) rates in CR and NR patients were 86.9%%, 55.5%, respectively.Conclusions: Our preliminary clinical results have shown that with current GIAC-3 protocol,the outcomes of haplo-BMT have been improved remarkably with lower aGVHD and better LFS and OS compared with our historic control. Haploidentical BM from the second donor as the third party cells is more feasible and cost effective compared with UCB. The mechanism of this strategy need to be further investigated. DisclosuresNo relevant conflicts of interest to declare.

Up-Front Randomization 10 Years Later: The AML Cooperative Group Strategy

As a global approach to acute myeloid leukemia the AMLCG started a clinical trial in 1999 with the intention to clarify the influence of baseline characteristics as well as treatment variables on patients’ outcome. The heterogeneity of this disease in mind, all randomization was done stratified for potential risk factors. Furthermore, all randomization was restricted to the single time point up-front of any treatment. Thus, an unbiased intention-to-treat analysis could be provided.Between June 1999 and January 2012, 3370 consecutive patients 16 - 86 (median 61) years of age entered the trial at 57 participating centers. The general up-front randomization comprised(A) repeated G-CSF priming versus no G-CSF,(B) double induction by TAD (thioguanine-araC-daunorubicin) all at standard dose plus HAM (high-dose araC- standard dose mitoxantrone) versus HAM plus HAM,(C) postremission TAD and maintenance versus TAD and autologous stem cell transplantation.For age adaption patients of 60 years and older received reduced intensity double induction (B) and maintenance instead of autologous transplantation (C).The complete remission rate was 57% (65% <60 and 51% 60+ years, p=<.0001). With a median observation time of 7.1 years the overall survival at 5 years is 26% (40% <60 years and 14% 60+ years, p<.0001) and the rate of patients in complete remission at 5 years is 29% (41% <60 years and 15% 60+ years, p<.0001). There were no significant differences in the CR rates, the 5 years survival and continuous complete remission rates according to randomizations for treatment variables.In contrast, the CR rates and the therapeutic endpoints projected to 5 years were found significantly influenced by risk factors as identified within both age groups and including favorable cytogenetics/ molecular genetics (Döhner H et al. Blood 115: 453-74; 2010), adverse genetics, AML secondary to cytotoxic treatment or to previous myelodysplastic syndrome, LDH in serum, and in particular increasing age itself.In patients between 16 and 59 years of age receiving equal intensity treatment those below versus above the median age of 48 years showed a CR rate of 69% versus 57% (p<.0001), a 5 years survival of 48% versus 33% (p<.0001), and a 5 years proportion in CR of 46% versus 35% (p<.0001). In patients between 60 and 86 years who received reduced intensity treatment the median age was 67 years, and those below versus above this median showed 51% versus 47% CR (p=.1), 5 years survival of 19% versus 9% (p<.001) and 19% versus 12% 5 years proportion in CR (p=.01).Thus, up-front randomization proved to provide unbiased informations about the impact of various risk factors and treatment variables.While age was found to dominate other risk groups (Büchner T et al. J Clin Oncol: 27: 61-69; 2009), a difference in treatment intensity by factor 3.7 had no influence of outcome in a sample of equal baseline characteristics (Büchner T et al. Blood 122: 1447; 2013). As an alternative to antileukemic cytotoxicity, allogeneic stem cell transplantation in first remission was found to prolong even the overall survival in a matched pair analysis again done prospectively within present AMLCG99 trial (Stelljes M et al. J Clin Oncol 32: 288-96; 2013).Up-front randomization and a common standard arm also qualified as a model to compare treatment strategies between different studies (Büchner T et al. Leukemia Research 26: 1073-75; 2002, J Clin Oncol 30:3604-10; 2012). In older patients a web-based prediction of complete remission and early death could be established based upon the AMLCG99 trial (Krug U et al. Lancet 376: 2000-08; 2010).In summary, present long-term results from up-front randomization strongly support a strict prospective strategy for cooperative clinical research on acute myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Effects of Priming with Recombinant Human Granulocyte Colony–Stimulating Factor on Conditioning Regimen for High-Risk Acute Myeloid Leukemia Patients Undergoing Human Leukocyte Antigen–Haploidentical Hematopoietic Stem Cell Transplantation: A Multicenter Randomized Controlled Study in Southwest China

HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective and immediate treatment for high-risk acute myeloid leukemia (HR-AML) patients lacking matched donors. Relapse remains the leading cause of death for HR-AML patients after haplo-HSCT. Accordingly, the prevention of relapse remains a challenge in the treatment of HR-AML. In a multicenter randomized controlled trial in southwestern China, 178 HR-AML patients received haplo-HSCT with conditioning regimens involving recombinant human granulocyte colony–stimulating factor (rhG-CSF) or non–rhG-CSF. The cumulative incidences of relapse and graft-versus-host disease (GVHD), 2-year leukemia-free survival (LFS), and overall survival (OS) were evaluated. HR-AML patients who underwent the priming conditioning regimen with rhG-CSF had a lower relapse rate than those who were treated with non-rhG-CSF (38.2%; 95% confidence interval [CI], 28.1% to 48.3% versus 60.7%, 95% CI, 50.5% to 70.8%; P < .01). The cumulative incidences of acute GVHD, chronic GVHD, transplantation-related toxicity, and infectious complications appeared to be equivalent. In total, 53 patients in the rhG-CSF–priming group and 31 patients in the non-rhG-CSF–priming group were still alive at the median follow-up time of 42 months (range, 24 to 80 months). The 2-year probabilities of LFS and OS in the rhG-CSF–priming and non-rhG-CSF–priming groups were 55.1% (95% CI, 44.7% to 65.4%) versus 32.6% (95% CI, 22.8% to 42.3%) (P < .01) and 59.6% (95% CI, 49.4% to 69.7%) versus 34.8% (95% CI, 24.9% to 44.7%) (P < .01), respectively. Multivariate analyses indicated that the 2-year probability of LFS of patients who achieved complete remission (CR) before transplantation was better than that of patients who did not achieve CR. The 2-year probability of LFS of patients with no M4/M5/M6 subtype was better than that of patients with the M4/M5/M6 subtype in the G-CSF–priming group (67.4%; 95% CI, 53.8% to 80.9% versus 41.9%; 95% CI, 27.1% to 56.6%; P < .05). This study suggests that the rhG-CSF–priming conditioning regimen is an acceptable choice for HR-AML patients, especially for the patients with no M4/M5/M6 subtype who achieved CR before transplantation.

Safety and feasibility of granulocyte colony-stimulating factor (G-CSF) primed bone marrow (BM) using three days of G-CSF priming as stem cell source for pediatric allogeneic BM transplantation.

There are limited data on the optimal dosing and schedule of G-CSF priming prior to BM harvest. We evaluated the safety and efficacy of three days of G-CSF of primed BM from related pediatric donors. Forty-five children were treated. All donors received 5 μg/kg per day of G-CSF as a single subcutaneous injection for three consecutive days prior to the BM harvest. The median age of the donors was seven yr (range, 0.8-18) and no donor experienced major adverse events related to G-CSF administration. The median age for the recipients was five yr (0.3-16 yr). Thirty-five patients had non-malignant disorders. The median dose of nucleated (TNC) and CD34+, CD3 cells infused per recipient weight was 5.4 × 10(8) /kg (range, 0.61-17), 4.7 × 10(6) /kg (range, 1.6-19), and 43.8 × 10(6) /kg (range, 1.8-95), respectively. All patients achieved neutrophil and platelets engraftment, at a median of 15 (range, 10-22) and 23 days (range, 13-111), respectively. At a median follow up of 60 months (range 12-100), the estimated five yr overall and EFS was 91% and 80%, respectively. Collection of BM following three days of G-CSF priming from pediatric donors is safe and results in high TNC and CD34+ cell yield.

Targeting Bone Marrow Lymphoid Niches In Acute Lymphoblastic Leukemia

Similar to normal lymphoid progenitors, acute lymphoblastic leukemia (ALL) blasts receive key growth and survival signals from the bone marrow microenvironment which contributes to chemotherapy resistance and disease relapse. We previously reported that treatment with G-CSF in mice results in a marked a marked suppression of lymphopoiesis by targeting lymphoid niches in the bone marrow (Day et al., ASH abstract #1246, 2012). Specifically, G-CSF suppresses the expression of lymphoid trophic factors from osteoblasts and CXCL12-abundant reticular (CAR) cells, including CXCL12, stem cell factor, and interleukin-7, and interleukin-6. We hypothesized that disruption of lymphoid niches in the bone marrow by G-CSF mediated suppression of lymphoid trophic factors will render ALL cells more sensitive to chemotherapy.We conducted a pilot study of G-CSF priming in patients with relapsed or refractory ALL (NCT01331590). Our preclinical data indicates that the timing of G-CSF in relation to chemotherapy is critical and that G-CSF must be given for at least 4 days prior to chemotherapy to achieve its maximal effect on the BM microenvironment. In this study, G-CSF (10 µg/kg/d) was administered continuously from day 1 until neutrophil recovery followed by ifosfamide 3330 mg/m2/d CIVI on days 4-6, etoposide 150 mg/m2 IV BID, and dexamethasone 5 mg/m2 BID on Days 4-10. Peripheral blood and BM samples are obtained at baseline and after 4 days of G-CSF (prior to chemotherapy). Where sufficient material was available, the baseline and day 4 G-CSF blood and BM samples were analyzed to assess: 1) changes in the BM microenvironment; 2) ALL cell mobilization into the blood; and 3) ALL cell proliferation and apoptosis. To assess expression of B trophic factors by BM stromal cells, we analyzed RNA from BM core biopsies at baseline and at day 4.Ten patients with with relapsed or refractory ALL with a median age of 47 years (range 21-69) have been enrolled. One subject developed leukocytosis to 67K/mm3 from baseline of 20K/mm3 requiring temporary cessation of G-CSF and earlier initiation of chemotherapy. One treatment related death occured on study. Three of 10 patients have achieved a CR/CRi (30%). Importantly, there were no cases of delayed hematologic recovery. Compared to healthy volunteers, the baseline expression of CXCL12, IL-7, and osteocalcin mRNA in the bone marrow was markedly variable, with some samples displaying very low expression. Nonetheless, G-CSF resulted in a 2-fold or greater decrease in CXCL12 expression in 6 of 7 cases tested. These data suggest that, although the BM microenvironment is disrupted at baseline in many patients with relapsed/refractory ALL, G-CSF can suppress lymphoid trophic factors even further. In 4 of 5 cases, mobilization of ALL blasts after G-CSF to the peripheral blood was observed (average fold-increase: 3.1 ± 0.95; range 1.3 to 6.2-fold). Interestingly, in the single case with minimal mobilization, little change in bone marrow expression of CXCL12 with G-CSF treatment was observed. ALL blast proliferation and apoptosis in the bone marrow was measured by flow cytometry before and after G-CSF priming. In all 3 samples that were analyzed, a significant increase in apoptosis, as measured by Annexin V surface expression, was observed [% Annexin V+ ALL blasts: 2.9 ± .81% (baseline) versus 13.2 ± 4.0% (G-CSF); p = 0.01]. Similar data were observed measuring apoptosis by activated caspase 3 expression. No change in ALL cell blast proliferation (as measured by Ki67 and DAPI staining) was observed.This study demonstrates the safety and feasibility of G-CSF priming in combination with chemotherapy in patients with relapsed or refractory ALL. Furthermore, G-CSF priming mobilizes ALL blasts into the blood and induces apoptosis these cells in the BM. Collectively, these data provide the proof of principle framework for ongoing studies targeting marrow lymphoid niches in ALL. [Display omitted] Disclosures:Off Label Use: sorafenib for AML.

Safety and Pharmacokinetics Of Clofarabine In Combination With High-Dose Cytarabine and Liposomal Daunorubicin In Pediatric AML: Results Of a Phase 1 Combination Study By The ITCC Consortium

IntroductionRelapsed/refractory pediatric AML has a poor prognosis despite salvage therapy including stem-cell transplantation. Chemotherapy using FLAG plus liposomal daunorubicine (FLAG-DNX) is currently considered the standard in 1strelapse in Europe (Kaspers et al, JCO 2013). FLAG is based on potentiation of cytarabine (Ara-C) by fludarabine (Flu) by increasing Ara-CTP levels. Clofarabine (CLO) is a novel purine nucleoside analog, designed to have improved efficacy. MethodsWe initiated an ongoing phase 1B dose-escalation study using a ‘3x3 design' to define the optimal dose of CLO, replacing FLU in FLAG-DNX. Dosages consisted of Ara-C 2gr/m2/day (day 1-5), with escalation of DNX from 40, 60 to 80 mg/m2/day (day 1, 3 and 5), and CLO from 20, 30 to 40 mg/m2/day (day 1-5) in 5 dose-levels, without GCSF priming. At day 6 intrathecal Ara-C was administered. Dose-level 1-4 recruited early 1st relapse, refractory 1st relapse and 2nd relapse of AML, and escalated CLO to 40 mg/m2 with DNX 60 mg/m2. Dose level 5 is open to 1st relapse pts and escalates DNX to 80 mg/m2. Responding patients received a 2ndconsolidation with CLARA-DNX or treated by investigator discretion. Other inclusion criteria consisted of performance status ≥60%, and normal liver and renal function. Maximum tolerated dose was the primary endpoint. Hematologic DLTs were defined as count recovery in responding pts >42 days. Serum and CSF were collected for pharmacokinetics (PK), taken at day 1: predose, T=2hrs and T=5 hrs post-infusion, and repeated on day 5, including a T=24 sample on day 6, together with a CSF sample just prior to intrathecal medication. CLO plasma and CSF concentrations were analyzed using LC-MS/MS. Efficacy data are awaiting central review and not released until the end of the study. The study is registered in the Dutch Trial Registry (nr. 1880). ResultsWe here report safety and PK data on the first 4 dose-levels after accrual of 22 AML patients (≥2nd relapse, n=7; refractory 1st relapse, n=9; early 1strelapse, n=6). Patients were treated at dose-level (DL) 1, n=4; 2, n=3; 3, n=12; 4, n=3. Eleven patients had been transplanted prior to enrollment. In total 27 CLARA-DNX courses were administered. Median age was 6.4 years, 14 patients were male. 20 SAEs were reported, consisting of febrile neutropenia (n=11), typhlitis (n=1), lung-infection fungal (n=3), lung-infection bacterial (n=2), candida sepsis (n=1), rash (hand-foot skin reaction) (n=1), and tumor lysis/capillary leak syndrome (n=1). Other grade 3-4 AEs reported in at least 2 patients consisted of abdominal pain, nausea and vomiting, diarrhea, anorexia and allergic reaction. Two patients had bilirubin elevation (max 61 umol/l); six patients had mild transaminase elevation > 100 U/l (max. 330U/l), which was reversible.At dose-level 3, two patients experienced DLTs (fungal infection) halting dose-escalation. Detailed analysis showed that most patients had evidence of subclinical fungal infections prior to enrollment. Therefore a protocol amendment was issued that required screening patients with CT-scan and serum-galactomannan prior to enrollment. After enrolling another cohort of 6 patients at DL3 this appeared to be safe (1 DLT of Candida sepsis out of 6 patients). No DLTs occurred at dose-level 4 in 3 patients. No hematological DLTs occurred.PK samples were available from 19 patients; in 2 patients sampling was repeated at course 2. At day 1 the median AUC was 28 ng/ml.mg.hr (range 6-401), with a mean T1/2 of 1.5 hrs. Day 1 and day 5 results were similar. CSF levels were not measurable in most patients and were 0.1-0.2 ng/ml.mg in the 3 patients with detectable levels. Our PK-data in combination are not significantly different from our data with single-agent CLO in relapsed ALL patients (Joel et al, AACR, 2007), and fit the CLO single agent PK model of Bonate et al (J Clin Pharmacol 2004, 44: 1309-32) . ConclusionsThe RP2D of CLO in a CLARA/DNX course in relapsed/refractory pediatric AML is 40 mg/m2, excluding patients with evidence of prior subclinical aspergillus. There is no evidence for PK interaction between CLO and the other drugs. We are currently testing the safety of an augmented dose of DNX (80 mg/m2) in 1strelapse AML patients. When tolerable this dose may be randomized against other induction regimens in front-line therapy in pediatric AML. AcknowledgmentThis study was financially supported by Sanofi and by the KiKa-foundation grant nr. 26. Disclosures:Off Label Use: Clofarabine for pediatric AML.