Clofarabine Research Articles

Benefits and risks of clofarabine in adult acute lymphoblastic leukemia investigated in depth by multi-state modeling.

We recently reported results of the prospective, open-label HOVON-100 trial in 334 adult patients with acute lymphoblastic leukemia (ALL) randomized to first-line treatment with or without clofarabine (CLO). No improvement of event-free survival (EFS) was observed, while a higher proportion of patients receiving CLO obtained minimal residual disease (MRD) negativity. In order to investigate the effects of CLO in more depth, two multi-state models were developed to identify why CLO did not show a long-term survival benefit despite more MRD-negativity. The first model evaluated the effect of CLO on going off-protocol (not due to refractory disease/relapse, completion or death) as a proxy of severe treatment-related toxicity, while the second model evaluated the effect of CLO on obtaining MRD negativity. The subsequent impact of these intermediate events on death or relapsed/refractory disease was assessed in both models. Overall, patients receiving CLO went off-protocol more frequently than control patients (35/168 [21%] vs. 18/166 [11%], p = 0.019; HR 2.00 [1.13-3.52], p = 0.02), especially during maintenance (13/44 [30%] vs. 6/56 [11%]; HR 2.85 [95%CI 1.08-7.50], p = 0.035). Going off-protocol was, however, not associated with more relapse or death. Patients in the CLO arm showed a trend towards an increased rate of MRD-negativity compared with control patients (HR MRD-negativity: 1.35 [0.95-1.91], p = 0.10), which did not translate into a significant survival benefit. We conclude that the intermediate states, i.e., going off-protocol and MRD-negativity, were affected by adding CLO, but these transitions were not associated with subsequent survival estimates, suggesting relatively modest antileukemic activity in ALL.

Older Adults with Acute Myeloid Leukemia (AML) Experience Improvement in Health-Related Quality of Life (HRQoL) Scores with Intensive Therapy: Prospective Study from ECOG-ACRIN (EA) E2906 Phase 3 Trial

Background There is limited prospective or comparative data evaluating the HRQoL impact of intensive chemotherapy (IC) in older adults (age ≥60 years) with AML. In the recent EA E2906 phase 3 study of '7&3' (daunorubicin & cytarabine) vs. clofarabine (CLO), a putative lower intensity therapy, we incorporated a prospective patient-reported outcomes (PRO) assessment of HRQoL and fatigue to understand treatment impact from the patients' (pts) perspective. Methods E2906 study design and results have been presented previously [n=727, med. age 68 years (range 60-86), randomized 1:1 to ‘standard’ ‘7&3‘ & high dose cytarabine (Arm A) vs. single agent CLO (Arm B), as remission induction (Step 1) and consolidation (Step 2), respectively]. There was no difference in composite complete remission (CCR, 50%) or 30-day mortality rates (8.5%), and in primary analysis CLO was inferior for Overall Survival. HRQoL was a key secondary protocol endpoint. PROs assessed HRQoL and leukemia-specific symptoms and concerns using Functional Assessment of Cancer Therapy-Leukemia scale (FACT-Leu), & Fatigue using FACT-Fatigue subscale. PROs were administered at prespecified time points: (1) Baseline; (2) Cycle 1 at nadir (approx. Day 15); (3) End of Step 1 restaging (approx. D 40); (4) Beginning of Step 2 (1 st consolidation, approx. D 45); and (5) 2 nd cycle consolidation/End Step 2 (approx. D 60). The FACT-Leu Trial Outcome Index ( TOI) was calculated by summing physical well-being, functional well-being, and leukemia-specific concerns subscales to create a single composite score. Higher TOI scores correspond to better HRQoL. The primary endpoint was to evaluate TOI difference from randomization to day 30 after induction therapy between arms, evaluated using Wilcoxon rank sum test. We also compared Clinically Meaningful differences (CMiD) in TOI (defined as patient change ≥½ standard deviation from baseline TOI) between arms, using the X2 test. Pts not completing at least 2 assessment forms (including baseline) were excluded. Testing of constructs of Physical Well-being (PWB), Functional, Leukemia & Fatigue scales demonstrated high Cronbach's a values (a >0.7) except for PWB (a=0.672) at a single time point (End of Step 2). Results Baseline TOI was available for n=489, and 364 pts (n=182 each Arm) were evaluable in this planned analysis. Evaluating CMiD during Cycle 1 induction, pts in Arm B were less likely to experience ‘Worse’ TOI score (32% vs. 40.7%, Arm A), more likely to maintain stable TOI scores (49.1% vs. 39%, Arm A), and had a similar proportion of ‘Better’ TOI scores (20.3%, A vs. 18.9%, B) although differences were not significant (p=0.1389). Conversely in cycle 1 consolidation, more patients in Arm A experienced Better TOI (45.1% vs. 29.2%, Arm B), fewer had stable TOI (45.1% vs. 58.3%, Arm B), and rates of ‘Worse’ TOI were similar (9.8%, A vs 12.5%, Arm B), again differences were not significant (p=0.2616). There was no difference in Fatigue scores between Arms (p=0.49). We evaluated whether TOI and Fatigue scores could predict the ability to complete protocol therapy. In univariate analysis baseline TOI (p=0.0059) and Fatigue (p=0.0057) were associated with Step 1 completion, however on multivariate analysis (adjusting for age, sex, and Arm) were no longer significant [TOI, Odds Ratio (OR) 1.03 (0.98-1.08, p=0.27); Fatigue, OR 1.03 (0.96-1.11, p=0.42)]. There was a borderline association of TOI with Step 2 completion [TOI OR 1.10 (1.00-1.23, p=0.06)]. Using a multivariable linear mixed effects model, we observed similar and significant changes in TOI scores in both Arms, demonstrating the effect of IC & CLO on HRQoL (Figure). Comparing to baseline, TOI scores decreased significantly at C1 induction nadir (-4.0873, p<0.0001), and increased by 5.0762 (p=0.0004) at end Step 1, by 6.9047 at cycle 1 consolidation (p<0.0001), and by 11.4601 (p<0.0001) at end Step 2. Differences were mainly apparent in those who achieved CCR, and there was significant attrition in PRO responses in non-CCR pts. Conclusions Older adults experienced significant improvement in HRQoL after nadir regardless of therapy, and those achieving CCR report significantly higher scores. We did not observe differences in leukemia specific HRQoL or fatigue between treatment Arms (7&3 vs. CLO). Patient-reported HRQoL & Fatigue was not predictive of completion of protocol therapy. These results serve as a benchmark for expectations with IC in older adults.

The HOVON 100 Study in Adult Acute Lymphoblastic Leukemia Re-Analysed By Multi-State Modeling: Benefits and Risks of Clofarabine

Background: Clofarabine (CLO) is an effective anti-leukemic drug that was recently evaluated in first line treatment of adult patients with acute lymphoblastic leukemia (ALL) in a prospective randomized study (HOVON 100). While more patients achieved eradication of measurable residual disease (MRD), no beneficial effect on the primary endpoint event-free survival (EFS) was noted (Rijneveld et al, 2022). To study these observations in more detail, we applied multi-state modeling to evaluate whether CLO affected intermediate events, including going to off protocol treatment (not due to relapse, but because of treatment-related toxicity) and achieving MRD negativity (defined as MRD<10-4), and the subsequent impact of these events on death or relapse. Methods: The HOVON 100 study randomized 334 adult ALL patients to the control (n=166) and to the CLO arm (n=168). Parameters determining disease risk were evenly distributed between the two study arms. Patients received induction and consolidation chemotherapy (Ind & Cons) followed by either allogeneic hematopoietic cell transplantation (alloHCT) or maintenance treatment, depending on risk status (irrespective of MRD), donor availability, and transplant eligibility. As recently reported, 5-year EFS was 50% (95%CI 42-57) in control patients compared with 53% (95%CI 45-60) in CLO treated patients. We developed a Markov time-inhomogeneous multi-state model describing the events between Ind & Cons (start state) until the date of last contact, incorporating maintenance treatment, off-protocol treatment, alloHCT, and relapse as intermediate events. Relapse mortality and non-relapse mortality were considered end states. We developed a second Markov time-inhomogeneous multi-state model that incorporated MRD negativity after consolidation as intermediate event, without considering off-protocol treatment. Patients with missing MRD data after consolidation (40%) were not considered MRD negative. All other states were evaluated as mentioned above. Effect estimation was done using a transition-specific Cox model including treatment arm (CLO vs control). Results: Overall, CLO treated patients went to off-protocol treatment almost as frequent as control patients (45/168 [27%] vs 36/166 [22%], P=0.31), but went to off-protocol treatment more frequently during maintenance treatment (13/44 (30%) vs. 6/56 (11%); HR 2.85 [1.08-7.50], P=0.035). Considering the treatment effect on the transitions to relapse, CLO treated patients had non-significantly different relapse during maintenance (HR 0.58 [0.24-1.39], P=0.22) than control patients. Such an effect was also not observed in alloHCT recipients (HR alloHCT to relapse: 0.86 [0.38-1.92], P=0.71) and was also not observed in patients who went to off-protocol treatment (HR 0.78 [0.31-1.98], P=0.60). With respect to MRD, patients in the CLO arm had a trend towards increased MRD negativity compared with control patients (HR Ind & Cons to MRD negativity: 1.36 [0.96-1.93], P=0.083). Additionally, MRD negative patients in the CLO arm were less likely to proceed to maintenance treatment (HR 0.45 [0.26-0.80], P=0.0065), did not proceed more often to alloHCT (HR 0.83 [0.50-1.38], P=0.46), and did not show less relapse than patients in the control arm (HR 1.08 [0.27-4.44], P=0.90). Transition probabilities of the multi-state "off-protocol” model per study arm are summarized in Figure 1. Conclusion: Multi-state modeling, taking going to off-protocol treatment and MRD negativity as intermediate events into account, identified that patients receiving CLO more often went to off-protocol treatment during maintenance, and that CLO treated patients less frequently proceeded to consolidation by alloHCT or maintenance treatment according to protocol. Despite a higher proportion of MRD negativity in patients receiving CLO, not completing protocol treatment might also explain similar relapse rates and EFS in the two arms. The addition of CLO to an already intensified chemotherapeutic schedule resulted in going to off-protocol treatment due to cumulative toxicities and not due to relapse for a higher proportion of patients. Future studies may evaluate CLO in a less intensified induction/consolidation/maintenance chemotherapeutic scheme, which differs from our pediatric inspired, intensified, backbone of the HOVON 100 ALL study. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clofarabin Added to Standard Treatment in Adult Patients with Newly Diagnosed ALL: First Results of the Randomized Phase III HOVON-100 Study

Background: clofarabine (CLO) is a nucleoside analogue with efficacy in acute myeloid and lymphoblastic leukemia (ALL). We evaluated whether CLO added to induction and consolidation would improve event free survival (EFS) in adult patients.Methods: patients, aged 18-70 years, with newly diagnosed precursor B- or T-ALL were eligible and randomized for standard treatment or combined with CLO. Standard treatment for younger (18-40 years) patients consisted of a pediatric inspired schedule (HOVON70, Rijneveld et al. 2011) and older patients (41-70 years) were treated with a semi-intensive schedule (HOVON71, Daenen et al. 2012). Patients with Ph positive ALL received imatinib during treatment, but not in combination with CLO. Patients were stratified according to age and immunophenotype (B- vs T-ALL). CLO (5 days, 30 mg/m2) was administered during 2 courses, i.e. during prephase with prednisone and as a single drug during a second consolidation course. The CLO dose was established during a run-in phase and was approved by the DSMB. High risk disease was defined as high WBC count at diagnosis, no CR after induction or high risk cytogenetic/molecular abnormalities (Ph or BCR-ABL positivity, MLL aberrations, hypodiploidy and complex karyotype). Allogeneic stem cell transplantation (alloSCT) with an HLA identical sibling donor was offered as consolidation to all patients and in patients with high-risk ALL a sibling or alternative donor was used. Primary endpoint was EFS. Secondary endpoints included CR, MRD measured by RQ-PCR of rearranged Ig or TCR genes, disease free (DFS) and overall survival (OS), toxicities and treatment related mortality. This trial was supported by Sanofi Genzyme and registered at www.trialregister.nl as NTR2004.Results: between December 2010 and November 2016, 340 patients were randomized. Here we report results of the first 120 younger and 120 older patients, i.e. 240 patients in total. No differences between study arms were observed with respect to pretreatment characteristics including age (median 41 year), risk group, Ph positivity, WHO-PS and immunophenotype. Respectively, 80% and 73% of patients ≤40 years completed protocol treatment according to arms A (control) and B (CLO); these percentages were 67% and 62% for patients >40 years. Estimated EFS was 49% and 46% at 5 years from randomization for arm A and B resp. (hazard ratio (HR): 1.05, 95% confidence interval (CI): 0.72-1.51, p=0.81; adjusted for age and phenotype). For patients ≤40 years, 5-year EFS was 58% vs 55% in arm A vs B, while in patients > 40 years EFS was 41% vs 29% in arm A vs B. CR rate was 87% and 88 % in arm A vs B and in both age groups. MRD was assessed at multiple time points during treatment and available for 117 (48%) patients, 58 and 58 in arm A and B resp. MRD response was defined by levels <10-4. 37/58 patients (64%) obtained MRD after consolidation 1 in arm A vs 49/59 (83%) in arm B (p=0.022). 49 (41%) of patients proceeded to alloSCT in arm A vs 54 (45%) in arm B (p=0.60). After a median follow up of 40 months (range: 1-72), 5-year OS was 59% in arm A vs 51% in arm B (HR = 1.17, 95% CI: 0.78-1.76, p=0.45). In patients who reached CR, 5-year DFS was 56% in arm A and 53% in arm B (HR = 1.08, 95% CI: 0.70-1.66, p=0.74), while 5-year relapse rates were 27% and 25% (HR = 1.03, 95% CI: 0.58-1.81, p=0.92), and 5-year non-relapse mortality was 17% vs 22 % (HR = 1.15, 95% CI: 0.59-2.25, p=0.69). No significant differences with respect to EFS, OS, and DFS emerged after evaluation with censoring of alloSCT recipients. Serious adverse events (SAEs) were slightly more often observed in CLO treated patients: 71% vs 82% in ≤40y (p=0.28) and 54% vs 65% in >40y (p=0.27). More infections grade 3-4 were observed in CLO treated patients: 44 vs 64% in arm A and B resp. (p=0.003).Conclusions: while complete evaluation of all 340 randomized patients is still ongoing, analysis of the first 240 randomized patients with sufficient follow-up (med 40 months) shows that addition of CLO is feasible. Toxicities included a non-significant increase of SAEs and a significant increase of infections. So far, a non-significant lower number of patients receiving CLO fully completed protocol treatment. Despite a higher MRD response in the CLO arm EFS, DFS and OS were not improved so far. Longer follow-up and analysis of all patients is required to determine whether a better MRD response translates into better outcome. [Display omitted] DisclosuresRijneveld:Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz pharmaceuticals: Research Funding. Petersen:Sanofi: Membership on an entity's Board of Directors or advisory committees. Selleslag:Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Schipperus:Amgen: Research Funding.

Gemcitabine/Clofarabine/Busulfan: A New Myeloablative Reduced-Toxicity Conditioning Regimen with High Activity for Allogeneic Stem-Cell Transplant (alloSCT) in Aggressive Lymphomas

INTRODUCTION:Stem-cell transplantation (SCT) is standard treatment of relapsed lymphomas. Patients in refractory relapse or relapsed after autoSCT are often considered for an alloSCT, aiming at a graft-vs.-lymphoma (GVL) effect. While reduced intensity conditioning regimens have substantially contributed safety to alloSCT, aggressive lymphomas often progress rapidly afterwards, partly due to tumor cell growth outpacing GVL. Outcomes are particularly poor for patients with active disease at the time of alloSCT. Thus, development of more efficacious conditioning regimens for aggressive lymphomas, avoiding mucotoxic drugs (such as melphalan), is a major need. We previously showed that the combination of 2 nucleoside analogues [gemcitabine (GEM) + clofarabine (CLO)] followed by busulfan (BU) is highly synergistic against B- and T-cell lymphoma cell lines, through inhibition of DNA damage repair. Those observations led us to clinically study a novel high-dose regimen of GEM/CLO/BU with alloSCT for aggressive lymphomas.METHODS:Eligibility included age 12-65; refractory DLBCL, T-cell and Hodgkin lymphomas; acceptable end-organ function, and an 8/8 HLA-matched (A, B, C and DRB1) sibling (MSD) or unrelated donor (MUD). GEM was escalated from dose level 1 to 3; it was given on days -6 (d-6) and d-4 as a loading dose of 75 mg/m2 followed by infusion at 10 mg/m2/min over 40-90 min (total daily dose 475, 675 or 975 mg/m2), to be followed by CLO (40 mg/m2/day x 4, d-6 to d-3) and BU (pharmacokinetically targeted to an average daily AUC of 4,000 μMol•min x 4, d-6 to d-3). AlloSCT was on d0. Patients receiving a MUD transplant received rabbit ATG (0.5, 1.5 and 2 mg/Kg on d-3, d-2, and d-1, respectively). Patients with CD20+ tumors received rituximab (375 mg/m2) on d-14, d-7, d+1 and d+8. The GVHD prophylaxis included tacrolimus (tapered after d+100) and mycophenolate mofetil (from d0 to d+60). Dose limiting toxicities (DLT) were defined as any G4-5 nonhematological organ toxicity, or as G3 skin or G3 mucositis lasting >3 days at peak severity. Dose escalation of gemcitabine followed a Bayesian design targeting a maximal DLT probability of 25%. Non-relapse mortality (NRM), relapse incidence, G II-IV or III-IV acute GVHD or chronic GVHD were calculated with the cumulative incidence method with consideration of competing risks.RESULTS:Between 11/12 and 06/17, 48 patients were enrolled: 25 DLBCL (5 double hit), 13 T-NHL and 10 HL (Table); 27 MSD, 20 MUD.All patients received PBPC. The MTD of gemcitabine was level 3. DLT was observed at the following frequencies: level 1: 12.5% (1/8), level 2: 9% (2/9), and level 3: 25% (4/16). Regimen-related toxicities were manageable, including mucositis (47% G2, 42% G3), self-limited transaminitis (21% G2, 49% G3), hyperbilirubinemia (no cases of VOD) (30% G2, 19% G3), and dermatitis (13% G2). There were no cardiac, pulmonary, renal or CNS toxicities. Neutrophils dropped to <0.1 in all patients on median d0 (d-3 to +2), and engrafted on median d+10 (d+9 to +20). Platelets engrafted on median d+12 (d+8 to +38).All patients engrafted; full donor chimerism was established early: a median of 100% (50-100%) on d+28, and 100% (98%-100%) on d+100. The rates of G II-IV and III-IV acute GVHD were 29.8% (35% MSD, 25% MUD) and 10.6% (19% MSD, 0% MUD), respectively. The rates of moderate-severe / severe chronic GVHD were 8.5% / 6.4%. 1/3 HL pts with prior exposure to anti-PD1 developed steroid-responsive hepatic chronic GVHD.The γ-H2AX levels in PBMNC (N=19) increased throughout treatment, to a median of 8.5 fold from baseline (range, 0.4-46.3) (P=0.0001) and PARP-1 decreased to 0.45 fold (0.2-6.3) (P=0.4), indicative of activation of DNA damage response and apoptosis, respectively. The overall response rates on d+30 (in 34 patients with measurable disease) are 77% (DLBCL), 100% (T-NHL), and 75% (HL). Their respective CR rates are 69%, 100% and 75%. The d+100 / 1-year NRM rates were 6.25% / 14.5%. NRM causes were infection (6) and GVHD (1). At median follow-up of 18 months (1-53) the EFS / OS rates of the entire population are 44% / 56%. Outcomes appear especially promising in the DLBCL (EFS / OS, 54% / 54%) and the T-NHL (62% / 69%) subgroups.CONCLUSIONS:GEM/CLO/BU is a new, myeloablative, reduced-toxicity alloSCT regimen with high activity in this population of heavily pretreated and refractory aggressive lymphomas, which warrants further study in disease-specific trials [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Minimal residual disease at end of induction and consolidation remain important prognostic indicators for newly diagnosed children and young adults with very high-risk (VHR) B-lymphoblastic leukemia (B-ALL): Children’s Oncology Group AALL1131.

10004 Background: Children and young adults with very high risk (VHR) B-acute lymphoblastic leukemia (B-ALL) [13-30 years of age with any features or 1-30 years of age with adverse prognostic features including KMT2A rearrangements, iAMP21, hypodiploidy (&lt;44 chromosomes/DNA index &lt; 0.81), central nervous system disease, end of induction (EOI) minimal residual disease (MRD) &gt;0.01%, or induction failure] collectively have a predicted 4-year disease free survival (DFS) of approximately 70%. Whether patients with VHR B-ALL who are MRD positive at EOI and become MRD negative at the end of consolidation (EOC) will have improved survival versus patients remaining MRD positive at EOC is unknown. Methods: Patients with newly diagnosed NCI high risk B-ALL enrolled on AALL1131 or NCI standard risk B-ALL enrolled on AALL0932 and classified as VHR at EOI were treated on the VHR stratum of AALL1131 which sought to improve DFS with intensive post-Induction therapy using fractionated cyclophosphamide (CPM), etoposide (ETOP) and clofarabine (CLOF).Patients were randomly assigned post-Induction to Control Arm (CA) with modified augmented BFM CPM + fractionated cytarabine + mercaptopurine, Experimental Arm 1 (Exp1) with CPM + ETOP, or Experimental Arm 2 (Exp2) with CLOF + CPM + ETOP during Part 2 of Consolidation and Delayed Intensification. Doses of vincristine and pegaspargase were identical on all arms. Exp2 was permanently closed September 2014 due to excessive toxicities, and these patients are excluded from this report. MRD was measured by 6-color flow cytometry at EOI and for those who consented at the EOC. Results: 4-yr DFS for all patients (n=823) with VHR B-ALL was 76.8 ± 2.0%. As we reported previously, 4-year DFS was not significantly different between CA and Exp 1 (85.5 ± 6.8% versus 72.3 ± 6.3%; p=0.76; Burke, Haematologica 2019). 4-yr DFS for patients who were EOI MRD &lt;0.01%, (n=325) versus &gt;0.01 (n=498) was 83.3% ± 2.6% vs 72.0% ± 2.8%, p=0.0013. 4-Year DFS of Patients EOI MRD &gt; 0.01%. Conclusions: MRD is a powerful prognostic indicator in VHR B-ALL with inferior outcomes in patients who are EOI MRD positive. Among patients who were EOI MRD positive treated on Exp1, outcomes were similar for EOC MRD negative and EOC MRD positive, though numbers were small. In contrast, patients who were EOI MRD positive treated on CA that were EOC MRD negative had significantly improved DFS compared to those that were EOC MRD positive. The CA remains the standard of care for COG ALL trials. With this therapy, patients with VHR B-ALL that are EOI MRD positive and EOC MRD negative have significantly improved DFS compared to those that remain MRD positive at EOC. Clinical trial information: NCT02883049. [Table: see text]

Exploring two-dimensional graphene and boron-nitride as potential nanocarriers for cytarabine and clofarabine anti-cancer drugs

Development in two-dimensional (2D) drug-delivery materials have quickly translated into biological and pharmacological fields. In this present work, pristine graphene (PG) and hexagonal boron nitride (h-BN) sheets are explored as a drug carrier for cytarabine (CYT) and clofarabine (CLF) anti-cancer drugs using density functional theory (DFT). The obtained geometrical, energetic and electronic properties revealed that the PG sheet is more reactive and it adsorbs CYT and CLF anti-cancer drugs better than the h-BN sheet. The adsorption energies of CYT and CLF on PG sheet is -24.293 and -23.308 kcal/mol respectively, this is due to the delocalized electrons present in the PG sheet. The flow of electron direction between anti-cancer drugs and 2D sheet are calculated by ΔN, ΔEA(B), and ΔEB(A) parameters and Natural bond orbital analysis (NBO). The electronic and optical properties are calculated to understand the chemical reactivity and stability of the complex systems. The obtained results exhibit that the PG sheet retains significant therapeutic potential as a drug delivery vehicle for a drug molecule to treat cancer therapy.

Ultralong-Circulating and Self-Targeting "Watson-Crick A = T"-Inspired Supramolecular Nanotheranostics for NIR-II Imaging-Guided Photochemotherapy.

A carrier-free theranostic nanodrug directly coassembled using a NIR probe and a chemotherapeutic drug is a promising alternative for cancer theranostics. Nevertheless, this nanodrug still faces the limitations of short blood circulation and inefficient tumor accumulation/tumoral cellular uptake in vivo. Meanwhile, most exogenous targeting ligands and poly(ethylene glycol) have no therapeutic effect. Herein, we designed an ultralong-circulating and self-targeting nanodrug by an ordered supramolecular coassembly of indocyanine green (ICG), methotrexate (MTX, chemotherapeutic drug and cancer-cell-specific ligand), and clofarabine (CA). Notably, CA, as a surfactant-like chemotherapeutic drug, was introduced into the initial ICG-MTX coassembly by "Watson-Crick A = T-inspired" hydrogen-bond-driven sequential assembly with MTX. This carrier-free theranostic nanodrug with exceptionally high drug payload (100 wt %) not only showed superior serum stabilities but also displayed ultralong blood circulation (>7 days), enabling efficient accumulation at tumor sites. Moreover, our nanodrugs could be self-recognized by cancer cells and release the drugs on demand through lysosomal acidity and external laser stimulus. Under NIR-II imaging guidance, high-efficiency tumor ablation via synergistic photothermal-chemotherapy could be achieved in one treatment cycle while preventing the tumor recurrence. Our ultralong-circulating and self-recognizing carrier-free theranostic nanodrug based on the "drug-delivering-drug" strategy might have the potential for clinical theranostic application.

Phase II trial with sequential clofarabine and cyclophosphamide for refractory and relapsed philadelphia-negative adult acute lymphoblastic leukemia. Results of the GIMEMA LAL 1610 protocol

Clofarabine (CLO) and cyclophosphamide (CY) combinations were tested in late stage refractory/relapsed (R/R) acute lymphoblastic leukemia (ALL) with disappointing results and high-grade toxicity. We designed a sequential 5-day combination of CLO 40 mg/m2/d plus CY 400 mg/m2/d as first salvage for Philadelphia-negative ALL patients refractory or relapsed until 24 months from complete remission (CR). Primary endpoint was an overall response rate (ORR) ≥ 40%. Among 26 study patients (median age 40.5 years) ORR was 57.6% (CR with complete [n = 8] or incomplete [n = 7] hematologic recovery). Despite severe myelotoxicity, no dose-limiting toxicity suggested de-intensification of CLO schedule. With a median follow-up of 17.0 months, median and 1-year overall and disease-free survival were 6.5 months and 28.6%, and 3.7 months and 28.1%, respectively. This association was tolerable and more effective in patients younger than 40 years with B-precursor ALL, longer first CR, not previously transplanted and achieving CR with full hematological recovery.

Tumor Microenvironment-Activated and Viral-Mimicking Nanodrugs Driven by Molecular Precise Recognition for dNTP Inhibition-Induced Synergistic Cancer Therapy.

The medical application of nanotechnology is promising for cancer chemotherapy. However, most of the small-molecule drug assemblies still have such disadvantages as serious drug leakage and nonideal synergistic mechanisms, resulting in undesired therapeutic effect. Both nucleoside analogue-based clofarabine (CA) and methotrexate (MTX) were used as the first-line anticancer medication. However, a single-agent chemotherapy still faced many challenges including low bioavailability and toxic side effects to normal tissues due to nonspecific biodistribution of drugs. Herein, we designed and fabricated novel viral-mimicking and carry-free nanodrugs (CA-MTX NPs) via molecular recognition-driven precise self-assembly between CA and MTX. After introduction of mild acid-responsive PEG-lipid on the surface of CA-MTX NPs, the synthetic nanodrugs with a diameter of ∼150 nm exhibited tumor microenvironment-activated characteristics and self-targeting property. The results suggested that our nanodrugs could achieve superior tumor accumulation and synergistically promote the tumor suppression by collaboratively inhibiting dNTP pools. We foresaw that the well-designed smart nanodrugs delivery system would open a new avenue in synergistic cancer therapeutics.

Minimal Residual Disease (MRD) at Time of Complete Remission Is Commonly Detected in Acute Myeloid Leukemia (AML) Patients Age ≥60 Years and Significantly Impacts Outcome Based on Post-Remission Treatment Strategies: Prospective Analysis of ECOG-ACRIN (E-A) E2906 Phase III Trial

Minimal Residual Disease (MRD) at Time of Complete Remission Is Commonly Detected in Acute Myeloid Leukemia (AML) Patients Age ≥60 Years and Significantly Impacts Outcome Based on Post-Remission Treatment Strategies: Prospective Analysis of ECOG-ACRIN (E-A) E2906 Phase III Trial

FLT3-ITD Mutations Are Prevalent and Significantly Impact Outcome after Intensive Therapy in Elderly Adults with Acute Myeloid Leukemia (AML): Analysis of the North American Intergroup E2906 Phase III Trial in Patients Age ≥60 Years

BackgroundActivating mutations in FLT3 - and in particular FLT3-ITD (internal tandem duplication) - are common in younger patients with AML, and are associated with relapse and with inferior overall survival (OS), however their prevalence and impact in older adults remains uncertain. We performed an analysis of FLT3-ITD mutations in patients age ≥60 yrs in the large prospective multicenter ECOG-ACRIN (E-A) E2906 Phase III trial.MethodsEligible patients (pts) age ≥60 yrs (n=727) were randomized to ‘Standard’ therapy with 7&3 (Dauno 60mg/m2) induction, and 2 cycles of intermediate dose Ara-C (1.5g/m2 x 12 doses; 6 doses if age ≥70yrs) consolidation (Arm A); or single agent clofarabine (CLO) induction and consolidation (2 cycles) (Arm B). As previously reported (Foran et al, ASH #217a, 2015), there was superior overall survival (OS) with standard therapy. AML diagnostic samples collected prospectively in the central E-A Leukemia Translational Research Lab (LTL) were used to detect mutations in the FLT3 gene by PCR using cDNA from total RNA. LTL investigators were blinded to treatment assignment. Patients with no RNA available or blast count <10% (threshold of sensitivity for the PCR assay) on the submitted sample were excluded. Statistical analysis was performed using X2 (categories) and Wilcoxon rank sum (continuous) tests to compare baseline patient and disease characteristics. Log-rank tests and multivariate Cox models stratified by treatment arm and adjusted for patient and disease variable (including WBC, cytogenetics, sex, performance status, secondary AML) were used to examine FLT3-ITD effect on OS and disease-free survival [DFS; relapse or death after complete remission (CR)/CRi (CR with incomplete CBC recovery)].ResultsIn the first 231 pts tested, FLT3-ITD mutations were identified in 43 (18.6%) pts, and the remainder were FLT3-ITD-negative (i.e. non-ITD). The proportion with FLT3-ITD was the same for patients age 60-69 yrs (18.8%) vs. ≥70 yrs (18.3%). In comparison to non-ITD, FLT3-ITD+ pts had significantly higher WBC (median 18.1 vs. 6.3, p=0.002) and BM blasts (80% vs. 51.5%, p=0.0004) at AML diagnosis, and were more likely to have intermediate risk (79.1% vs. 59%) vs. unfavorable risk (7.0 % vs. 31.4%) cytogenetics (p=0.002). There was no difference in CR/CRi rate overall (p=0.40), however standard (Arm A) pts with FLT3-ITD had a significantly higher CR/CRi rate (78.9% vs. non-ITD 51.5%, p=0.04). With median follow-up 53.5 months, FLT3-ITD patients tended to have worse OS (HR 1.24, 95%CI 0.85-1.81) but this was not statistically significant (Figure 1, p=0.26), and results were similar for both Arms A/B. DFS similarly tended to be worse overall for FLT3-ITD (HR 1.44, 95%CI 0.65-2.43) (p=0.17), and DFS was significantly worse for FLT3-ITD+ Arm A/Standard pts (Figure 2, p=0.033), while Arm B/CLO pts had a worse DFS regardless of FLT3 mutation status (Arm B, FLT3-ITD vs. non-ITD, p=0.93). More patients with FLT3-ITD underwent allogeneic transplantation (25.6% vs. non-ITD 19.7%), although this was not significant (p=0.41).ConclusionsFLT3-ITD mutations are prevalent in older and elderly (age ≥70 yrs) patients with AML, and while they occur at somewhat lower rates than reported for younger adults, FLT3-ITD+ AML in this population has a leukemia phenotype similar to that reported in younger patients. Despite significantly higher CR/CRi rates with Standard therapy, older pts with FLT3-ITD also have significantly worse DFS following intensified Ara-C consolidation therapy than non-ITD pts. These results support routine assessment of FLT3-ITD status in older AML patients, and the incorporation of novel post-remission treatment strategies to improve outcome in FLT3-ITD+ pts ≥60 yrs. [Display omitted] DisclosuresForan:Agios: Research Funding; Xencor, Inc.: Research Funding. Lazarus:Pluristem Ltd.: Consultancy. Tallman:BioSight: Other: Advisory board; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; Cellerant: Research Funding; Orsenix: Other: Advisory board; ADC Therapeutics: Research Funding; AROG: Research Funding.

Targeting Ribonucleotide Reductase M1 and M2 in Multiple Myeloma

Ribonucleotide reductase (RR) is an enzyme that catalyzes the conversion of ribonucleotide diphosphate to deoxyribonucleotide diphosphate and is essential for de novo DNA synthesis, DNA repair, and cell growth. RR primarily exists as a heterodimer tetramer composed of regulatory subunit RRM1, and catalytic subunit RRM2. Overexpression or polymorphisms of RR are described non-small cell lung, pancreas, breast, and ovarian cancers. Moreover, RRM1 and RRM2 expression is highly correlated with patient survival in non-small cell lung and pancreas cancers. To date, the biologic significance in multiple myeloma (MM) has not yet been elucidated. In this study, we characterized the role of RRM1 and RRM2 in MM pathogenesis.By examining 3 independent expression data from GEO database, we found that RR (especially RRM1) expression is higher in MM plasma cells than normal plasma cells. We examined RRM1 and RRM2 expression in 6 MM cell lines and 3 MM patients' tumor cells by quantitative real-time PCR and immunoblotting and confirmed that both RRM1 and RRM2 are highly expressed in these cells. We next knocked down both RRM1 and RRM2 in 4 MM cell lines (NCI-H929, MM.1S, RPMI8226, and KMS-11) using siRNA. Knockdown of RRM1 and RRM2 triggered significant growth inhibition, associated with apoptotic cells death evidenced by AnnexinV/PI staining, in NCI-H929 and MM.1S, but not RPMI8226 or KMS-11 cells. We next examined molecular mechanisms whereby RRM1 downregulation triggers MM cell death. Gene expression profiling showed that p53 regulated genes were overexpressed after RRM1 knockdown. We therefore further examined DNA damage response (phosphorylated-ATM, -ATR, -Chk1, and -Chk2, and gamma-Histone H2A.X) and p53 (phosphorylated-p53, p21, Noxa, Puma, Bax) signaling pathways and found that these pathways were activated in NCI-H929 and MM.1S (both p53 wild-type), but not in RPMI8226 (p53 mutant) or KMS-11 (p53 null) cells after RRM1 knockdown. To validate the role of RRM1 of in vivo, we subcutaneously injected MM.1S cells transduced with shRNA against RRM1 or shLuc into our mouse xenograft model and observed that tumor growth was significantly reduced in shRRM1-MM.1S cells versus shLuc-MM.1S cells. Clofarabine (CLO), a purine nucleoside analog, which allosterically inhibits both DNA polymerases and RRM1 is used to treat acute leukemia and chronic lymphocytic leukemia and has been studied preclinically in MM (Valdez et. al. Experimental Hematology 2013). We therefore next examined CLO as a potential therapeutic agent in MM. Consistent with RRM1 knockdown, CLO induced growth arrest in p53 wild-type cell lines (NCI-H929, MM.1S, and MOLP-8), but not in p53 mutant (RPMI8226, OPM2, U266) or null (KMS-11) cells. Moreover, CLO treatment combined with DNA damaging agents (Melphalan, Doxorubicin) triggered synergetic cell death in p53 wild-type MM cells.Our results therefore demonstrate that RR, especially RRM1, is a novel therapeutic target in patients with wild-type p53 MM, and provide the basis for clinical evaluation of CLO, alone or in combination with DNA damaging agents, to improve patient outcome. DisclosuresHideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Quantification of Newer Anti-Cancer Drug Clofarabine in their Bulk and Pharmaceutical Dosage Form

Quantitative determination of Clofarabine in their Pharmaceutical Dosage form was carried out by Liquid chromatographic method. The aim of the present work was to develop a RP-HPLC method for Clofarabine (CFB). The column used was Luna C-18 column (250 mm × 4.6 mm id, 5 m particle size) using Water: Methanol (60:40 v/v) as a mobile phase at a flow rate of 1.0 ml/min. Quantification was achieved with UV detection at 266 nm over the concentration range 0.05-20 μg/ml and % recovery was found to be in the range of 99.58-100.95 for CFB by the RPHPLC method. The proposed method was validated with respect to linearity, accuracy, precision, selective, sensitive and robustness. The method was fruitfully applied for the estimation of clofarabine in injection dosage forms.

North American Leukemia, Intergroup Phase III Randomized Trial of Single Agent Clofarabine As Induction and Post-Remission Therapy, and Decitabine As Maintenance Therapy in Newly-Diagnosed Acute Myeloid Leukemia in Older Adults (Age ≥60 Years): A Trial of the ECOG-ACRIN Cancer Research Group (E2906)

▪Background: Induction therapy with daunorubicin (Dauno) & cytarabine (Ara-C) [DA] has been the standard of care for eligible older adults (age ≥ 60 years) with newly diagnosed acute myeloid leukemia (AML) for over 2 decades. Single agent Clofarabine (CLO) induction & consolidation (Consol.) therapy has demonstrated important clinical activity in this age group in large phase II studies. Lower induction mortality (IM) & similar reported complete remission rate (CR) & overall survival (OS), as well as notable activity in those with higher risk disease features [including unfavorable cytogenetics, therapy-related AML (t-AML) & prior antecedent hematologic disorder (AHD)] raises the possibility that a non-Ara-C-based regimen could achieve similar or superior OS with lower toxicity.Methods: We performed a randomized United States Intergroup Phase III trial of single agent CLO [30mg/m2 x 5 days induction; 20 mg/m2 re-induction (if indicated) & 2 cycles Consol.] vs. standard DA therapy [Dauno 60mg/m2 D1-3 & Ara-C 100mg/m2 D1-7 induction x 1-2 cycles; 2 cycles Consol. with Ara-C (1.5g/m2 Q12hrs D1-6 age 60-69; once daily if age 70+)] in patients (pts) age ≥ 60 yrs with newly diagnosed AML. Patients with serum creatinine >1.0 (or GFR <60 mL/min) and those with AML-M3 and ECOG performance status >3 (PS>2 if age 70+ yrs) were excluded. Randomization was stratified by age (60-69 vs. 70+), t-AML, & AHD. Pts with HLA-matched donor were eligible for allogeneic transplantation (AlloHCT) after induction, and those completing Consol. were eligible for randomization #2 (R#2) to maintenance decitabine [20mg/m2 x 3D, monthly x 1 year] versus observation. With a target accrual of 747, E2906 was powered to determine non-inferiority [and possible superiority] of CLO vs. standard DA, and primary endpoint was OS. A weighted statistical analysis was performed to account for confounding impact of R#2. AlloHCT patients were censored at transplant in this analysis. Responses & cytogenetics were confirmed centrally and OS & CR rates were monitored by an independent Data Safety Monitoring Committee (DSMC) at pre-specified time points.Results: As of Feb 23, 2015, 727 pts were randomized. Median age was 68 years (range 60-86); 57% were male, and 38% were age ≥70 yrs. Treatment arms are well balanced for all baselineclinical & AML characteristics, & 30% had unfavorable cytogenetics. Of 659 with complete treatment information reported, 30.4% on DA vs. 40.1% on CLO received 2 cycles of induction (p=0.006).Median follow-up of surviving patients is 7.6 months.Table 1shows early treatment results (CR, toxicity) for the 686 pts randomized as of Dec 23, 2014 (2 months prior to study end, & excluding 90 with ongoing response evaluation).DACLOp-valueCR/CRi43.8%42.8%p=0.8730-day mortality8.5%7.9%p=0.8960-day mortality14.9%13.1%p=0.58Gr 4-5 Non-Heme Tox.Induction27%19%p=0.02Gr 4-5 Non-Heme Tox.Consol.20%7%p=0.001374 pts have died (174, DA; 200, CLO) & significantly inferior OS was observed for CLO vs. DA [Hazard Ratio (HR) 1.41 (95% CI 1.12-1.78)] (Fig. 1). Planned subgroup analyses were performed (Table 2) demonstrating significant differences in OS after CLO for patients age 60-69 yrs, without AHD, & with intermediate risk cytogenetics; but not for those with Unfav. Cytogen. (Fig. 2) or t-AML. Based on the primary weighted analysis, DSMC recommended suspension of new accrual to E2906 on Feb 23, 2015 & all active patients on CLO were transitioned to DA Arm. Table 2NHR CLO/Standard (95% CI)*Weighted OS7271.41 (1.12-1.78)Unweighted OS7271.23 (1.00-1.50)Age 60-694491.48 (1.10-1.99)Age 70+2781.34 (0.93-1.93)Intermed. Risk Cytogen.3781.77 (1.27-2.47)Unfav. Risk Cytogen.2160.96 (0.65-1.43)No AHD6041.46 (1.13-1.89)AHD1231.22 (0.74-2.00)De novo AML6271.52 (1.18-1.96)Therapy-related AML1000.94 (0.54-1.61)Conclusions: Despite similar CR & IM, OS after single agent CLO is inferior to standard DA therapy for pts age ≥60 years with newly diagnosed AML who are fit for intensive therapy, and DA remains the standard of care. However no difference in OS was observed after CLO in some pre-specified high risk AML subgroups. R#2 & AlloHCT arms continue in E2906 for pts already enrolled. Embedded prospective minimal residual disease study at CR is being performed to identify pts at higher risk after CLO & DA. [Display omitted] [Display omitted] DisclosuresOff Label Use: Use of clofarabine in AML, and maintenance therapy with decitabine in AML. Claxton:Medimmune: Research Funding; BMS: Consultancy; Astellas: Research Funding; Cyclacel: Research Funding; Merck: Research Funding; Ambit: Research Funding. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy. Altman:Seattle Genetics: Consultancy; BMS: Consultancy; Spectrum: Consultancy; Astellas: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Al-Kali:Novartis: Research Funding.

Feasibility of intensive post-Induction therapy incorporating clofarabine (CLOF) in the very high risk (VHR) stratum of patients with newly diagnosed high risk B-lymphoblastic leukemia (HR B-ALL): Children’s Oncology Group AALL1131.

10007 Background: Four-year disease free survival (DFS) for patients with B-ALL classified as VHR is approximately 70%. Strategies to improve DFS with intensive post-Induction therapy using fractio...

Oligomeric Regulation of Ribonucleotide Reductase (RNR) by Antileukemic Nucleotides

Human RNR (hRNR) is the rate‐limiting enzyme for the de novo dNTP synthesis required for DNA replication and repair. hRNR activity is positively correlated with cancer cell proliferation and nucleotide analogs that can suppress hRNR activity such as gemcitabine and clofarabine (ClF) are proven effective in chemotherapy. However, the precise mechanisms by which many of the nucleoside prodrugs inhibit hRNR are unknown in most cases. The active hRNR is minimally an α2β2 holocomplex. Leukemia drug ClF is shown to inhibit hRNR through persistent hexamerization of α (α6) without requiring β. We hypothesize that α hexamerization is a universal mechanism by which antileukemic nucleotides inhibit hRNR, ultimately presenting a powerful small‐molecule approach to regulate hRNR. To test this hypothesis, I have sought to establish the mechanisms of hRNR inhibition by two additional clinically used nucleoside prodrugs—cladribine (CLA) and fludarabine (FLU). My studies have revealed that di‐ and triphosphates are active forms of both drugs that reversibly inhibit hRNR. CLA nucleotides [CLAD(T)P] are more potent than FLU nucleotides [FLUD(T)P] (Ki= 0.8, 0.1, 4.9 and 6.6 µM, respectively). In all cases, enzyme inhibition is coupled to α6 formation. We further validate in NIH‐3T3 cells stably expressing wild type α and hexamerization‐defective mutant α that hRNR inhibition by these nucleosides is directly correlated with oligomeric deregulation in cellular environment.

Sulforaphane Alone and in Combination with Clofarabine Epigenetically Regulates the Expression of DNA Methylation-Silenced Tumour Suppressor Genes in Human Breast Cancer Cells

Background/Aim: Sporadic breast cancer is frequently associated with aberrant DNA methylation patterns that are reversible and responsive to environmental factors, including diet. In the present study, we investigated the effects of sulforaphane (SFN), a phytochemical from cruciferous vegetables, on the methylation and expression of PTEN and RARbeta2 tumour suppressor genes as well as on the expression of regulators of DNA methylation reaction, DNMT1, p53, and p21, in MCF-7 and MDA-MB-231 human breast cancer cells with different invasive potential. We also evaluate the role of SFN epigenetic effects in support of therapy with clofarabine (ClF) that was recently shown to modulate the epigenome as well. Methods: Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis and real-time PCR, respectively. Results: In both MCF-7 and MDA-MB-231 cells, SFN at IC₅₀ (22 and 46 µ<smlcap>M</smlcap>, respectively) and a physiologically relevant 10 µ<smlcap>M</smlcap> concentration lead to hypomethylation of PTEN and RARbeta2 promoters with concomitant gene up-regulation. The combination of SFN and ClF enhances these effects, resulting in an increase in cell growth arrest and apoptosis at a non-invasive breast cancer stage. Conclusions: Our findings provide evidence that SFN activates DNA methylation-silenced tumour suppressor genes in breast cancer cells and may contribute to SFN-mediated support of therapy with an anti-cancer drug, ClF, increasing its applications in solid tumours.

Clofarabine in Combination with High-Dose Cytarabine and Liposomal Daunorubicin in Pediatric AML: Results of a Phase 1B Combination Study By the ITCC Consortium

Introduction: Relapsed/refractory pediatric AML has a poor prognosis. Chemotherapy using FLAG plus liposomal daunorubicine (FLAG-DNX) followed by stem-cell transplantation (SCT) is currently considered the standard in 1strelapse in Europe (Kaspers, JCO 2013), and ~results in ~40% survival. FLAG is based on potentiation of high-dose cytarabine (Ara-C) by fludarabine (Flu) by increasing Ara-CTP levels. Clofarabine (CLO) is a novel purine nucleoside analog, designed to have improved efficacy.Methods: We initiated a phase 1B 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 to 60 mg/m2/day (day 1, 3 and 5), and CLO from 20, 30 to 40 mg/m2/day (day 1-5) in 4 dose-levels (DL). At day 6 intrathecal Ara-C was administered. Patients with early 1st relapse, refractory 1st relapse and 2nd relapse of AML were eligible. Responding patients received a consolidation with CLARA-DNX or treated by investigator discretion. DL 5 was open for patients with early 1st relapse only, and dose-escalated DNX to 80 mg/m2. MTD was the primary endpoint. At the MTD the cohort was expanded to 10 patients. Hematologic DLTs were defined as count recovery in responding pts >42 days. Overall response rate (ORR) was defined as: partial remission (PR, 50% reduction BM blasts), no evidence of leukemia (NEL, <5% blasts, no regeneration), complete remission with insufficient recovery (CRi, platelets below 80/μl and neutrophils below 1000/μl) and complete remission (CR). Morphology was centrally reviewed by the AML-BFM SG in Hannover. The study is registered in the Dutch Trial Registry (nr. 1880), and was open in 14 ITCC/I-BFM-sites.Results: DL 1-4 accrued 29 AML patients (≥2nd relapse, n=8; refractory 1st relapse, n=11; early 1strelapse, n=10). Patients were treated at dose-level (DL) 1, n=4; 2, n=3; 3, n=12 (split in 3A and 3B); 4, n=10. Twelve patients had been transplanted prior to enrollment. In total 38 CLARA-DNX courses were administered. Median age was 8.8 years, 18 patients were male, median WBC at enrollment 8.7x10.9/l. 26 SAEs were reported, consisting of febrile neutropenia (n=14), typhlitis (n=1), lung-infection fungal (n=3), lung-infection bacterial (n=2), candida sepsis (n=1), streptococcal sepsis (n=2), rash (hand-foot skin reaction) (n=1), and tumor lysis/capillary leak syndrome (n=1), cardiovascular failure (n=1) and vomiting (n=1). Other grade 3-4 AEs reported consisted of abdominal pain, nausea and vomiting, diarrhea, anorexia. blood pressure changes, DIC, rash and renal failure. Three pts had bilirubin elevation (>2.5xULN, max 61 µmol/l); 8 transaminitis (> 100 U/l; max. 998U/l), 1 elevated creatinine (83µmol/l) and 1 renal failure, 5 low potassium (<3 mmol/l) and 3 low sodium (<130mmol/l). At DL 3A, DLTs (fungal infection) halted dose-escalation. Detailed analysis showed subclinical fungal infections prior to enrollment. A protocol amendment was issued requiring screening patients with CT-scan and serum-galactomannan. After enrolling DL3B this appeared to be safe (1 DLT of Candida sepsis out of 6 patients). One DLT occurred at dose-level 4 (sepsis). Time to recovery of peripheral blood values in patients with CR/CRi was median 30 days for neutropenia and 26 days for platelets.Considering outcome, the ORR was 59% in the 26 pts evaluable for response. In the 11 pts with refractory 1st relapse after FLAG the ORR was 38%. In the expansion cohort (10 pts in DL4) there was 1 CR, 7 CRi, 1 PR, and 1 early death. 18 pts were transplanted post clofarabine treatment (1 HLA-id, 13 MUD, 1 MFD, 2 haplo, 1 unkown). The median follow-up time of the 9 survivors was 16 months. The 1-year overall survival was 48±9%, and the 1-year EFS was 33±9%.Five patients were recruited in DL5. Two DLTs occurred in 2 pts, i.e. a severe pseudomonas cellulitis and a Gram-negative sepsis, and hence this cohort was closed.Conclusions: The RP2D of CLO in a CLARA/DNX course in relapsed/refractory pediatric AML is 40 mg/m2 combined with DNX 60 mg/m2 and Ara-C 2 gr/m2, excluding patients with evidence of prior subclinical fungal infection. This combination resulted in a high response rate in the expansion cohort. We observed responses in 38% of patients refractory to FLAG. This regimen therefore has greater anti-leukemic potential than FLAG.Acknowledgment: This study was financially supported by Sanofi, and by the KiKa-foundation grant nr. 26. DisclosuresZwaan:Sanofi: Research Funding. Off Label Use: Clofarabine is off-label for AML, and so is Daunoxome.

Simultaneous determination of fludarabine and clofarabine in human plasma by LC-MS/MS.

A method for quantification of fludarabine (FDB) and clofarabine (CFB) in human plasma was developed with an API5000 LC-MS/MS system. FDB and CFB were extracted from EDTA plasma samples by protein precipitation with trichloroacetic acid. Briefly, 50 μL plasma sample was mixed with 25 μL internal standard (50 ng/mL aqueous 2-Cl-adensosine) and 25 μL 20% trichloroacetic acid, centrifuged at 25,000 × g (20,000 rpm) for 3 min, and then transfered to an autosampler vial. The extracted sample was injected onto an Eclipse extend C18 column (2.1 mm×150 mm, 5 μm) and eluted with 1mM NH4OH (pH 9.6) - acetonitrile in a gradient mode. Electrospray ionization in positive mode (ESI(+)) and multiple reaction monitoring (MRM) were used, and ion pairs 286/134 for FDB, 304/170 for CFB and 302/134 for the internal standard were selected for quantification. The retention times were typically 3.72 min for FDB, 4.34 min for the internal standard, 4.79 min for CFB. Total run time was 10 min per sample. Calibration range was 0.5-80 ng/mL for CFB and 2-800 ng/mL for FDB. The method was applied to a clinical pharmacokinetic study in pediatric patients.