Administration Of Fludarabine Research Articles

A Tender Reduced-Intensity Conditioning for the Unfit: A Novel 4 Gy Total Body Irradiation-Based Conditioning Followed by Two-Step Haploidentical Stem Cell Transplant, Results of a Prospective Trial

Allogeneic hematopoietic stem cell transplant (HSCT) remains the only potentially curative treatment for many hematologic malignancies (HM). We previously developed a two-step approach that separates the lymphoid and myeloid portions of the graft, allowing a consistent T cell dosing and sparing the stem cells from the effect of post-transplant cyclophosphamide (CY). The two-step approach demonstrated safety and efficacy in patients treated with myeloablative and reduced-intensity conditioning. Here, we extended our two-step platform to older and less fit patients and explored the effects of using a high dose of T cells on disease relapse and transplant outcomes. Thirty-four patients with HM were treated. Median age was 68 years old and included a minority population constituting 32%. Eighty-two percent had a hematopoietic cell transplantation comorbidity index score ≥3. Ninety-one percent were haploidentical, and the rest were matched-related donor HSCT. Following administration of fludarabine and 2 Gy total body irradiation (TBI) (13 patients) or 4 Gy TBI (21 patients) conditioning regimen, a fixed dose of 2 × 108/kg CD3+ T cells was given, followed 2 days later by CY, then infusion of CD34-selected stem cells. Overall survival (OS) was 70% at 1 year and 48% at 3 years. The cumulative incidence (CI) of non-relapse mortality (NRM) and relapse were 22% and 33% at 3 years. However, the CI of relapse was much lower for patients treated with 4 Gy TBI versus those treated with 2 Gy TBI (11% versus 54%, P = .045), while NRM was similar (23% versus 15%, P = .399). This contributed to a high OS of 64% in patients who received 4 Gy TBI-based conditioning at 3 years, with median OS not reached, although this was not statistically significant (P = .68). The median time to neutrophil and platelet recovery was 12 and 17 days, respectively. The CI of grade II acute graft-versus-host-disease (aGVHD) was 22% and 26% at 100 days and 6 months, respectively. The CI of chronic GVHD (cGVHD) was 7.5% at 3 years. There was no grade III or IV aGVHD, no severe cGVHD, and no deaths attributable to GVHD. In conclusion, the two-step approach HSCT demonstrated a low disease relapse rate and high survival in patients treated with 4 Gy TBI-based conditioning, despite a generally older and more medically compromised patient population.

Fludarabine-induced bradycardia in allogeneic hematopoietic stem cell transplantation: A retrospective study.

Fludarabine, a purine analog, is getting more attention with the increasing use of reduced intensive conditioning regimens in allogeneic hematopoietic stem cell transplantation (allo-HSCT). The side effect of bradycardia was observed in only a few cases reported in the literature. In clinical practice, bradycardia can be asymptomatic or cause syncope and cardiac arrest. This study aimed to evaluate the bradycardia side effect of fludarabine used in the conditioning regimen in allo-HSCT recipients and to increase awareness of this issue. This retrospective study included 73 patients who received fludarabine in the allo-HSCT conditioning regimen between January 2015 and January 2021. Patients with and without bradycardia were compared regarding demographic data, allo-HSCT characteristics, electrolyte values, fludarabine administration dose and duration, and survival. Univariate and multivariate analyzes were performed to evaluate independent predictors for fludarabine-induced bradycardia. Fludarabine administration doses and days were higher in the bradycardia group, but no statistically significant difference was observed. In the multivariate analysis, age was the only independent predictor of fludarabine-induced bradycardia (odds ratio (OR) 0.93, 95% confidence interval (CI): 0.89-0.98, p = 0.007). The median age in the group with bradycardia was 19 years younger than those without bradycardia (34 (19-49) vs 53 (19-69), p = 0.005). In 11 (84.6%) of the patients who had bradycardia, bradycardia improved with the discontinuation of fludarabine alone, but atropine was administered in 2 (15.4%) patients. Age was the only independent predictor of fludarabine-induced bradycardia; therefore, close heart rate monitoring is recommended during fludarabine administration, especially in younger patients.

STAT1 Contributes to Microglial/Macrophage Inflammation and Neurological Dysfunction in a Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) triggers a plethora of inflammatory events in the brain that aggravate secondary injury and impede tissue repair. Resident microglia (Mi) and blood-borne infiltrating macrophages (MΦ) are major players of inflammatory responses in the post-TBI brain and possess high functional heterogeneity. However, the plasticity of these cells has yet to be exploited to develop therapies that can mitigate brain inflammation and improve the outcome after TBI. This study investigated the transcription factor STAT1 as a key determinant of proinflammatory Mi/MΦ responses and aimed to develop STAT1 as a novel therapeutic target for TBI using a controlled cortical impact model of TBI on adult male mice. TBI induced robust upregulation of STAT1 in the brain at the subacute injury stage, which occurred primarily in Mi/MΦ. Intraperitoneal administration of fludarabine, a selective STAT1 inhibitor, markedly alleviated proinflammatory Mi/MΦ responses and brain inflammation burden after TBI. Such phenotype-modulating effects of fludarabine on post-TBI Mi/MΦ were reproduced by tamoxifen-induced, selective KO of STAT1 in Mi/MΦ (STAT1 mKO). By propelling Mi/MΦ away from a detrimental proinflammatory phenotype, STAT1 mKO was sufficient to reduce long-term neurologic deficits and brain lesion size after TBI. Importantly, short-term fludarabine treatment after TBI elicited long-lasting improvement of TBI outcomes, but this effect was lost on STAT1 mKO mice. Together, our study provided the first line of evidence that STAT1 causatively determines the proinflammatory phenotype of brain Mi/MΦ after TBI. We also showed promising preclinical data supporting the use of fludarabine as a novel immunomodulating therapy to TBI.SIGNIFICANCE STATEMENT The functional phenotype of microglia and macrophages (Mi/MΦ) critically influences brain inflammation and the outcome after traumatic brain injury (TBI); however, no therapies have been developed to modulate Mi/MΦ functions to treat TBI. Here we report, for the first time, that the transcription factor STAT1 is a key mediator of proinflammatory Mi/MΦ responses in the post-TBI brain, the specific deletion of which ameliorates neuroinflammation and improves long-term functional recovery after TBI. We also show excellent efficacy of a selective STAT1 inhibitor fludarabine against TBI-induced functional deficits and brain injury using a mouse model, presenting STAT1 as a promising therapeutic target for TBI.

Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice.

Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics.

Long-term follow-up after lymphodepleting autologous haematopoietic cell transplantation for treatment-resistant systemic lupus erythematosus.

Autologous haematopoietic cell transplantation (AHSCT) improves immunologic dysfunction in patients with SLE. However, the curative potential of this therapy remains uncertain. This study reports outcomes in SLE patients receiving a lymphodepleting, reduced intensity regimen for AHSCT in SLE. Eight patients with SLE refractory to treatment, including i.v. cyclophosphamide (CYC), were enrolled. Five had LN and three CNS involvement as primary indications for transplant. Haematopoietic cell mobilization with CYC, G-CSF and rituximab was followed by collection of CD34+ positively selected cells. The conditioning regimen consisted of concurrent administration of CYC, fludarabine and rituximab. All immunosuppressive medications were discontinued at the start of mobilization and CS were rapidly tapered after the transplant. Five of eight patients achieved a complete response, including a decline in the SLEDAI to zero, which was sustained in four patients for a median of 165 months (range 138-191). One patient achieved a partial response, which was followed by relapse at month 18. Two patients with nephritis and underlying comorbidities in most organs had early deaths from infection and multiorgan failure. AHSCT resulted in profound lymphodepletion, followed by expansion of Treg cells and repopulation of naive T and B cells. Patients with a complete response showed a sustained suppression of the SLE-associated IFN-induced gene signature, marked depletion of memory and plasmablast B cells and resultant sustained elimination of anti-dsDNA antibody. Durable clinical and serologic remissions with suppression in the IFN gene signature can be achieved in refractory SLE following lymphodepleting AHSCT. ClinicalTrials.gov, https://clinicaltrials.gov, NCT00076752.

Determinants for Oral Mucositis in Allogeneic Hematopoietic Stem Cell Transplantation: Prospective Evaluation and Risk Analysis

Background: Oral mucositis (OM), secondary to mucosal barrier injury of pre-hematopoietic stem cell transplantation (HSCT) conditioning, is a common and painful toxicity of transplantation. Mucositis has been associated with increased risk for infections, hospitalization, analgesia and total parenteral nutrition use, and higher costs. However, prevalence and incidence data for mucositis are inconsistent and underreported. We set to prospectively identify risk factors for oral mucositis in patients undergoing allogeneic HSCT.Methods: OM was prospectively evaluated on a weekly basis in patients undergoing allogeneic HSCT. The grade of OM was determined based on the National Cancer Institute Common Toxicity Criteria for Adverse Events (CTCAE) scale (v. 4.0). Severe OM was defined as grade 2 or higher. Conditioning regimens were evaluated individually and according to intensity; myeloablative (MAC), reduced intensity (RIC) or reduced toxicity (RTC). The latter category included only patients receiving Fludarabine and Treosulfan (Flu/Treo). Variables associated with severe OM (p<0.05) were initially identified by univariate analysis (Chi-square and Mann Whitney U tests) and then analyzed in a multivariate logistic regression model. Cox and Fine and Gray regression models were used to study the association between severe OM and overall survival or nonrelapse mortality, respectively.Results: A total of 95 patients who underwent an allogeneic HSCT from 2016 to 2017 were included. Median age was 61 (range 19-75). Leading indications for HSCT were acute myeloid leukemia (51%), followed by myelodysplastic syndrome (15%), and non-Hodgkin lymphoma (12%). The majority of patients had a comorbidity score ≥3 (51%), were exposed to systemic antibiotics preceding transplantation (53%), and had no history of recent smoking (86.%). Donors were primarily HLA matched unrelated (51.1%) or HLA matched siblings (40%). RIC, MAC, and RTC regimens were used in 41%, 34%, and 25% of the patients, respectively. Methotrexate (MTX) (72.6%) or Mycophenolate mofetil (MMF) (27.4%), in conjunction with Cyclosporine, were given for graft versus host disease (GVHD) prophylaxis. Median follow-up was 165 days (95% CI 130-200).Median OM grade was 2 (range 0-4), with 65% of the patients experiencing severe mucositis. Median time from HSCT to OM and duration of OM were 8 days (1-14) and 10 days (2-40), respectively. Patients receiving Flu/Busulfan (Bu)-4 days (Flu/Bu4) were more likely to develop severe OM (91% vs. 9%, p<0.01), while among those conditioned with Flu/Treo, 29% had severe OM vs. 71% who did not (p<0.01) (Figure). In a univariate analysis, considering a broad range of patient, disease, and transplant characteristics (Table), only a history of recent smoking (19% vs. 3%, p=0.028), exposure to antibiotics in the preceding month pre-HSCT (61% vs. 39%, p=0.049), and use of MTX for GVHD prophylaxis (85% vs. 49., p<0.001) were associated with severe mucositis. Patients receiving RTC (i.e. Flu/Treo conditioning) had lower rates of severe OM compared to both RIC and MAC (p<0.001). Pre-HSCT albumin levels, obesity, and a low estimated glomerular filtration rate (eGFR) were not associated with OM. In a multivariate logistic regression model use of MTX vs. MMF had an increased risk for severe OM (odds ratio [OR] 11.6 [95% CI 3.2-42.4], p<0.001); compared to MAC, the risk for severe OM was reduced with RTC (OR 0.1 [0.0-0.4], p=0.001. OR for recent smoking was 6.8 (0.7-64.5), with a trend toward statistical significance (p=0.09). Severe OM was not associated with a decrease in short term overall survival (HR 0.59 [95% CI 0.2-1.5] or increase in non-relapse morality (HR 0.51 [0.1-2]).Conclusion: Our findings suggest that administration of Fludarabine and Treosulfan is an appealing option for conditioning since the risk for oral toxicity is low while intensity is high. Though having a borderline statistical significance, peri-transplantation smoking emerges as an important risk factor for oral mucositis. Therefore, patient counseling about smoking could potentially lower rates of mucositis. In contrast to previous reports, obesity and pre-HSCT albumin and reduced eGFR were not associated with mucositis in our cohort. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Similar Survival but Increased Toxicity with a Sequential Versus Concurrent FluBu4 Regimen

Combined in vitro treatment with fludarabine and busulfan has been shown to lead to synergistic cytotoxicity. Maximal synergy is achieved when fludarabine is administered immediately prior to busulfan. However, fludarabine and myeloablative busulfan (FluBu4) has been administered in both a concurrent and a sequential, non-overlapping fashion. Because timed sequential regimens are becoming increasingly popular, we sought to compare survival and relapse outcomes as well as toxicity between patients receiving allogeneic stem cell transplant (SCT) with a concurrent 5-day versus a sequential 9-day FluBu4 regimen.We analyzed 102 consecutive patients who received FluBu4 prior to allogeneic SCT between 2003 and 2016. Thirty-seven (36%) patients received the 5-day regimen, which consisted of fludarabine 40 mg/m2 and intravenous busulfan (target AUC 4800 mmol-min per dose) administered daily from Day -5 to Day -2. In 65 (64%) patients, the 9-day regimen was administered, utilizing equivalent doses of fludarabine on day -9 to day -6 and busulfan on day -5 to day -2. All patients received graft-versus-host disease (GVHD) prophylaxis with tacrolimus and methotrexate. Patients undergoing a matched unrelated donor transplant received anti-thymocyte globulin.Baseline characteristics including gender, race, median time to transplant, median lines of prior treatment, disease risk index, hematopoietic cell transplantation-comorbidity index (HCT-CI), donor type, stem cell source, and mean CD34 count were similar between the two groups. Patients in the 9-day group were younger compared to the 5-day group (median age 45 versus 51 years, p=0.003). The majority of patients in both groups were diagnosed with acute myeloid leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia.The median time to neutrophil and platelet engraftment was not different between the two groups. More patients receiving the 9-day regimen experienced Grade 4 mucositis (53 versus 6 patients, p<0.0001). Thus, 23 (35.4%) patients in the 9-day group versus 5 (13.5%) patients in the 5-day group required a day +11 methotrexate dose reduction or omission (p=0.02). This resulted in a higher cumulative incidence of both Grade 2-4 (27% versus 38%, p=0.01) and Grade 3-4 (11% versus 34%, p=0.006) acute GVHD (aGVHD) in the 9-day group compared to the 5-day group. In a multivariate analysis, receiving the 9-day regimen predicted for a greater risk of grade 3-4 aGVHD (p=0.025). Additonally, extensive chronic GVHD was found to be higher in the 9-day FluBu4 cohort (19% vs. 40% p=0.047).Median one-year GVHD-free, relapse-free survival (GRFS) was significantly worse for patients receiving a 9-day FluBu4 regimen (68.4 versus 144 days, p=0.032). In a multivariate analysis adjusted for HCT-CI, a trend towards significance was observed with the 5-day regimen predicting for longer GRFS compared to the 9-day regimen (p=0.07). Notably, we observed no differences in overall survival, progression-free survival, or relapse rates between the 5- and 9-day regimen.This is the first study to compare outcomes between a concurrent 5-day and sequential 9-day FluBu4 regimen. We demonstrate similar survival and relapse rates, suggesting that concurrent administration of fludarabine and busulfan may not be necessary for synergistic cytotoxicity. At the same time, we observed increased early toxicity, less MTX administration, and higher aGVHD rates in patients receiving sequential 9-day FluBu4. Contrary to what has been observed with timed sequential regimens, our data suggests that a prolonged administration schedule of conditioning chemotherapy leads to greater toxicity resulting in higher GVHD rates. DisclosuresPatel:Celgene: Consultancy, Honoraria.

Azeliragon ameliorates Alzheimer’s disease via the Janus tyrosine kinase and signal transducer and activator of transcription signaling pathway

OBJECTIVES:TTP488, an antagonist of the receptor for advanced glycation end-products, was evaluated as a potential treatment for patients with mild-to-moderate Alzheimer’s disease (AD). However, the mechanism underlying the protective action of TTP488 against AD has not yet been fully explored.METHODS:Healthy male rats were exposed to aberrant amyloid β (Aβ) 1-42. Lipopolysaccharide (LPS) and the NOD-like receptor family pyrin domain containing 1 (NLRP1) overexpression lentivirus were injected to activate the NLRP1 inflammasome and exacerbate AD. TTP488 was administered to reverse AD injury. Finally, tofacitinib and fludarabine were used to inhibit the activity of Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) to prove the relationship between the JAK/STAT signaling pathway and TTP488.RESULTS:LPS and NLRP1 overexpression significantly increased the NLRP1 levels, reduced neurological function, and aggravated neuronal damage, as demonstrated by the impact latency time of, time spent by, and length of the platform covered by, the mice in the Morris water maze assay, Nissl staining, and immunofluorescence staining in rats with AD.CONCLUSIONS:TTP488 administration successfully reduced AD injury and reversed the aforementioned processes. Additionally, tofacitinib and fludarabine administration could further reverse AD injury after the TTP488 intervention. These results suggest a new potential mechanism underlying the TTP488-mediated alleviation of AD injury.

Efficiency and safety of autologous chimeric antigen receptor T-cells therapy used for patients with lymphoma: A systematic review and meta-analysis.

Chimeric antigen receptor (CAR) T-cell therapy has produced promising response rates in patients with B cell malignancies. However, previous meta-analyses have demonstrated that CAR T-cell efficacy is unsatisfactory in patients with lymphoma unlike in patient with other hematological malignancies, but these studies included insufficient numbers of studies and patients with lymphoma. Furthermore, clinicians are interested in the effects of infusion dose, CAR structure, interleukin-2 (IL-2), and conditioning therapy regimen. All clinical trials administering autologous CAR T-cell therapy in lymphoma patients were searched in medical databases. A traditional meta-analysis was performed to assess the safety and efficacy of CAR T-cells in lymphoma treatment. Subgroup analysis was performed to determine the relationships between potential factors and efficacy. The best overall response rate (ORR), 6 month ORR (6m ORR), and severe cytokine release syndrome (sCRS) rate were calculated by Stata 14.0. A total of 411 patients across all the studies were included. Our analysis showed a best ORR of 0.71, a 6m ORR of 0.63, and an overall CRS (grade ≥ 3) rate of 0.18. The subgroup analysis showed that increased response rates and reduced CRS (grade ≥ 3) rates were associated with a low dose of CAR T-cells. No IL-2 administration and the use of a fludarabine-containing lymphodepletion regimen led to improved efficacy, while anti-CD19 CAR T cells led to a more successful outcome than anti-CD20 CAR T cells. In addition, 2nd- and 3rd-generation CAR T cells exhibited increased effectiveness in clinical studies, and no significant effect diversity was found between the 2nd- and 3rd-generation CAR T cells. sCRS was associated with a high dose of infused CAR T cells when IL-2 and fludarabine were excluded from the positive factors for sCRS. CAR T cells are promising in the treatment of relapsed or refractory lymphoma. Doses lower than 10/m, no IL-2 administration, fludarabine administration, and anti-CD19 CAR T cells were related to improved efficacy and safety.

Fludarabine as a cost-effective adjuvant to enhance engraftment of human normal and malignant hematopoiesis in immunodeficient mice

There is still an unmet need for xenotransplantation models that efficiently recapitulate normal and malignant human hematopoiesis. Indeed, there are a number of strategies to generate humanized mice and specific protocols, including techniques to optimize the cytokine environment of recipient mice and drug alternatives or complementary to the standard conditioning regimens, that can be significantly modulated. Unfortunately, the high costs related to the use of sophisticated mouse models may limit the application of these models to studies that require an extensive experimental design. Here, using an affordable and convenient method, we demonstrate that the administration of fludarabine (FludaraTM) promotes the extensive and rapid engraftment of human normal hematopoiesis in immunodeficient mice. Quantification of human CD45+ cells in bone marrow revealed approximately a 102-fold increase in mice conditioned with irradiation plus fludarabine. Engrafted cells in the bone marrow included hematopoietic stem cells, as well as myeloid and lymphoid cells. Moreover, this model proved to be sufficient for robust reconstitution of malignant myeloid hematopoiesis, permitting primary acute myeloid leukemia cells to engraft as early as 8 weeks after the transplant. Overall, these results present a novel and affordable model for engraftment of human normal and malignant hematopoiesis in immunodeficient mice.

439 - Similar Survival but Increased Toxicity with a Sequential Versus Concurrent FluBu4 Regimen

Abstract Combined in vitro treatment with fludarabine and busulfan has been shown to lead to synergistic cytotoxicity. Maximal synergy is achieved when fludarabine is administered immediately prior to busulfan. However, fludarabine and myeloablative busulfan (FluBu4) has been administered in both a concurrent and a sequential, non-overlapping fashion. Because timed sequential regimens are becoming increasingly popular, we sought to compare survival and relapse outcomes as well as toxicity between patients receiving allogeneic stem cell transplant (SCT) with a concurrent 5-day versus a sequential 9-day FluBu4 regimen. We analyzed 102 consecutive patients who received FluBu4 prior to allogeneic SCT between 2003 and 2016. Thirty-seven (36%) patients received the 5-day regimen, which consisted of fludarabine 40 mg/m2 and intravenous busulfan (target AUC 4800 mmol-min per dose) administered daily from Day -5 to Day -2. In 65 (64%) patients, the 9-day regimen was administered, utilizing equivalent doses of fludarabine on day -9 to day -6 and busulfan on day -5 to day -2. All patients received graft-versus-host disease (GVHD) prophylaxis with tacrolimus and methotrexate. Patients undergoing a matched unrelated donor transplant received anti-thymocyte globulin. Baseline characteristics including gender, race, median time to transplant, median lines of prior treatment, disease risk index, hematopoietic cell transplantation-comorbidity index (HCT-CI), donor type, stem cell source, and mean CD34 count were similar between the two groups. Patients in the 9-day group were younger compared to the 5-day group (median age 45 versus 51 years, p=0.003). The majority of patients in both groups were diagnosed with acute myeloid leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia. The median time to neutrophil and platelet engraftment was not different between the two groups. More patients receiving the 9-day regimen experienced Grade 4 mucositis (53 versus 6 patients, p Median one-year GVHD-free, relapse-free survival (GRFS) was significantly worse for patients receiving a 9-day FluBu4 regimen (68.4 versus 144 days, p=0.032). In a multivariate analysis adjusted for HCT-CI, a trend towards significance was observed with the 5-day regimen predicting for longer GRFS compared to the 9-day regimen (p=0.07). Notably, we observed no differences in overall survival, progression-free survival, or relapse rates between the 5- and 9-day regimen. This is the first study to compare outcomes between a concurrent 5-day and sequential 9-day FluBu4 regimen. We demonstrate similar survival and relapse rates, suggesting that concurrent administration of fludarabine and busulfan may not be necessary for synergistic cytotoxicity. At the same time, we observed increased early toxicity, less MTX administration, and higher aGVHD rates in patients receiving sequential 9-day FluBu4. Contrary to what has been observed with timed sequential regimens, our data suggests that a prolonged administration schedule of conditioning chemotherapy leads to greater toxicity resulting in higher GVHD rates. Disclosures Patel: Celgene: Consultancy, Honoraria.

The analysis of the parameters of 24-hr ECG Holter monitoring in patients with blood neoplasms undergoing high-dose chemotherapy and stem cell transplantation.

Hematopoietic stem cell transplantation (HSCT) is a widely used procedure in the treatment of malignant diseases, including blood neoplasms and has increased survival in hematological diseases. The aim of the study was to analyze parameters of 24-hr ECG monitoring in patients with selected blood neoplasms in whom the procedure of hematopoietic stem cell transplantation was performed. The study group consisted of 64 adults diagnosed with hematologic cancer qualified for HSCT with the previous high dose chemotherapy (HDC). In all patients 24-hr Holter monitoring was carried out twice. First examination took place prior to the HSCT procedure, and the second after finishing the procedure of HSCT. The minimal and mean heart rate (HR min and HR max) from 24-hr ECG recording was statistically significantly higher after the transplantation in comparison with the first test. The number of premature ventricular complexes (PVCs) was higher in the test after HSCT. In the second examination there was significantly higher percentage of premature ventricular complexes, incidents of tachycardia, and Mobitz type 1 second degree atrioventricular block. In regression analysis, in a group of patients with blood neoplasms after HSCT and HDC, administration of cyclophosphamide, fludarabine and total body irradiation were independent risk factors for electrocardiographic abnormalities in 24-hr Holter monitoring, that is, the increase in HR min, HR mean and PVCs. In patients with blood neoplasms undergoing HSCT more electrocardiographic abnormalities may be found after this procedure in comparison with the 24-hr Holter monitoring before transplantation.

Fludarabine, Cytarabine, Daunoxome Plus Dasatinib Has High Efficacy with an Acceptable Toxicity Profile As Either Consolidation or Salvage Regimen in Adult Philadelphia Positive Acute Lymphoblastic Leukemia Patients

BACKGROUND AND AIMSThe prognosis of Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL) patients has improved since the introduction of tyrosine kinase inhibitors (TKI). The inclusion of TKIs in standard ALL protocols allows a great increase in complete molecular responses, but at the price of non negligible toxicities and high rates of toxic deaths. On the other and TKI monotherapy as induction treatment allows to rapidly achieve complete hematologic remission (CR) but only a minority of patients achieve a complete molecular response with high risk of relapse. On the other hand,In the last years we tested a combination of Fludarabine, Cytarabine, Daunoxome (FLAD) with or without TKIs (mainly Dasatinib) as salvage regimen in relapsed-refractory ALL, with acceptable toxicity and good efficacy. We decided to apply the same schedule in newly diagnosed Ph+ ALL as consolidation treatment after a two months TKI (Dasatinib) monotherapy induction on a minimal residual disease condition.MATERIALS AND METHODSFLAD regimen consisted with a three-days administration of Fludarabine 30 mg/sqm followed four hours later by Cytarabine 2000 mg/sqm and Daunoxome 100 mg/sqm. TKI were suspended during chemotherapy administration and were re-administrated starting from day 5. G-CSF was given to all patients from day 4 to complete hematological recovery. FLAD was administrated for up to two cycles; all patients with available donor proceeded to allogeneic bone marrow transplantation (allo-BMT) after FLAD. Minimal residual disease (MRD) was evaluated in all patients after each FLAD either by RQ-PCR for VDJ rearrangements, multicolor flow cytometry (MFC) and RQ-PCR for BCR/Abl.Ten Ph+ ALL have been treated with FLAD + TKIs from January 2008 to December 2014: six patients received FLAD as salvage regimen, two of them in post allo-BMT setting, whereas four patients were treated frontline, after hematological CR was obtained with Dasatinib + steroids induction. All frontline patients proceeded to allo-BMT after two FLAD. Median age for frontline patients was 50 years (range 29-58), median follow-up was 20 months.RESULTSAs salvage regimen, 5/6 patients achieved hematological CR after FLAD, with three patients achieving also MFC MRD negativity and clearance of VDJ and BCR/Abl transcript. All patients who did not receive subsequent BMT relapsed, whereas of the two transplanted patients one is still in CR after a follow-up of 38 months.In the frontline setting, all patients received 70 days induction of Dasatinib + Steroids and achieved CR with complete hematological recovery. BCR/Abl transcript could be detected in all patients on BM samples on day 33 and on day 70 (Fig. 1), two patientshad MFC MRD positivity both on day 33 and on day 70, whereas two patients achieved MFC MRD negativity on day 33.FLAD was very well tolerated, with negligible non hematological toxicity, with a median duration of ANC <500 and PLT <20000 of 7 and 9 days, respectively, slightly higher in the second course. Median time between the beginning of first and second course was 35 days, whereas median time from second course to allo-BM was 44 days.Two patients achieved BCR/Abl negativity after first FLAD. All patients achieved molecular complete response after the second course (Fig. 1). No patient experienced relapse, whereas one patient died in CR on day +289 after allo-BMT due to myocardial viral infection.CONCLUSIONSFLAD has a very good efficacy in adult Ph+ ALL, with an acceptable toxicity profile. Deep responses have been observed in relapsed patients, and all newly diagnosed patients who received FLAD as consolidation regimen had achieved molecular CR before allo-BMT. Achieving complete hematological response with Dasatinib + steroids allowed us to safely administer two FLAD courses. [Display omitted] DisclosuresOff Label Use: Use of liposomal daunorubicin in the treatment of ALL.

Severe fludarabine neurotoxicity after reduced intensity conditioning regimen to allogeneic hematopoietic stem cell transplantation: a case report

Key Clinical MessageWe present a case of severe, irreversible neurotoxicity in a 55-year-old-patient with myelofibrosis undergoing hematopoietic stem cell transplantation following a reduced intensity conditioning including fludarabine. The patient developed progressive sensory-motor, visual and consciousness disturbances, eventually leading to death. MRI imaging pattern was unique and attributable to fludarabine neurotoxicity.

Association of fludarabine pharmacokinetic/dynamic biomarkers with donor chimerism in nonmyeloablative HCT recipients.

Fludarabine monophosphate (fludarabine) is an integral component of many reduced-intensity conditioning regimens for hematopoietic cell transplantation (HCT). Fludarabine's metabolite, 9-β-D-arabinofuranosyl-2-fluoroadenine (F-ara-A), undergoes cellular uptake and activation to form the active cytotoxic metabolite fludarabine triphosphate (F-ara-ATP), which inhibits cellular DNA synthesis in CD4(+) and CD8(+) cells. In this study, we evaluated whether fludarabine-based pharmacologic biomarkers were associated with clinical outcomes in HCT recipients. Participants with hematologic diseases were conditioned with fludarabine and low-dose total body irradiation (TBI) followed by allogeneic HCT and post-grafting immunosuppression. After fludarabine administration, we evaluated pharmacological biomarkers for fludarabine-F-ara-A area under the curve (AUC) and the ratio of circulating CD4(+) and CD8(+) cells (CD4(+)/CD8(+) ratio) after fludarabine administration-in 102 patients; F-ara-ATP accumulation rate in enriched CD4(+) and CD8(+) cells was evaluated in 36 and 34 patients, respectively. Interpatient variability in the pharmacological biomarkers was high, ranging from 3.7-fold (F-ara-A AUC) to 39-fold (F-ara-ATP in CD8(+) cells). Circulating CD8(+) cells were more sensitive to fludarabine administration. A population pharmacokinetic-based sampling schedule successfully allowed for estimation of F-ara-A AUC in this outpatient population. There was a poor correlation between the F-ara-AUC and the F-ara-ATP accumulation rate in CD4(+) (R (2)=0.01) and CD8(+) cells (R (2)=0.00). No associations were seen between the four biomarkers and clinical outcomes (day +28 donor T cell chimerism, acute graft-versus-host disease (GVHD), neutrophil nadirs, cytomegalovirus reactivation, chronic GVHD, relapse, non-relapse mortality, or overall mortality). Considerable interpatient variability exists in pharmacokinetic and fludarabine-based biomarkers, but these biomarkers are not associated with clinical outcomes in fludarabine/TBI-conditioned patients.

Endothelial Function in Patients with Hematologic Malignancies Undergoing High-Dose Chemotherapy Followed by Hematopoietic Stem Cell Transplantation

The aim of the study was to examine endothelial function in patients with hematological malignancies treated with high-dose chemotherapy followed by hematopoietic stem cell transplantation. The studies were conducted on 43 consecutive patients qualified for HSCT following high-dose chemotherapy based on the current standards. Then, due to exclusion criteria, a group of 38 patients were chosen for further investigations. Evaluation of endothelial function by means of flow-mediated dilatation (FMD) was conducted in patients with hematological malignancies before HSCT (test A) and after HSCT (test B). Brachial artery diameter (BAD) after occlusion, change in BAD and FMD were significantly lower after HSCT as compared to the results obtained before the transplantation (p < 0.05). The regression analysis indicated that administration of fludarabine and cytarabine, and also higher blood concentrations of creatinine represented risk factors for the impairment of endothelial function expressed as decreased FMD value. In patients with hematopoietic malignancies treated with HSCT, endothelial function assessed by the flow-mediated dilatation was impaired after chemotherapy and stem cell administration.

AMPK Suppresses Vascular Inflammation In Vivo by Inhibiting Signal Transducer and Activator of Transcription-1.

Activation of AMPK suppresses inflammation, but the underlying mechanisms remain poorly understood. This study was designed to characterize the molecular mechanisms by which AMPK suppresses vascular inflammation. In cultured human aortic smooth muscle cells, pharmacologic or genetic activation of AMPK inhibited the signal transducer and activator of transcription-1 (STAT1), while inhibition of AMPK had opposite effects. Deletion of AMPKα1 or AMPKα2 resulted in activation of STAT1 and in increases in proinflammatory mediators, both of which were attenuated by administration of STAT1 small interfering RNA or fludarabine, a selective STAT1 inhibitor. Moreover, AMPK activation attenuated the proinflammatory actions induced by STAT1 activators such as interferon-γ and angiotensin II (AngII). Mechanistically, we found that AMPK activation increased, whereas AMPK inhibition decreased, the levels of mitogen-activated protein kinase phosphatase-1 (MKP-1), an inducible nuclear phosphatase, by regulating proteasome-dependent degradation of MKP-1. Gene silencing of MKP-1 increased STAT1 phosphorylation and prevented 5-aminoimidazole-4-carboxyamide ribonucleoside–reduced STAT1 phosphorylation. Finally, we found that infusion of AngII caused a more severe inflammatory response in AMPKα2 knockout mouse aortas, all of which were suppressed by chronic administration of fludarabine. We conclude that AMPK activation suppresses STAT1 signaling and inhibits vascular inflammation through the upregulation of MKP-1.

Fludarabine and bradycardia in recipients of allogeneic stem cell transplant: a case series

Fludarabine is being increasingly used both as a chemoimmunotherapeutic agent and in conditioning regimens for stem cell transplant. ECG disturbances with this increasingly used drug have never been reported. This report describes a patient who developed bradycardia following fludarabine administration, prompting an assessment for rhythm irregularities in subsequent patients undergoing stem cell transplant using a fludarabine-based conditioning regimen. In a series of 10 patients who received fludarabine as part of their conditioning, there was a significant effect on QTc interval (mean difference 30.6 ms; P = 0.002) and heart rate (mean difference −18.9 beats/min; P = 0.003). In conclusion, caregivers need to be alert to recognize the potential for unrecognized adverse events even in frequently used drugs. Fludarabine has a potential for rhythm disturbances.

Fludarabine, cytarabine and Idarubicin (FLAI-5) with or without Gemtuzumab Ozogamicin Is an Effective and Well Tolerated Regimen for Younger AML Patients: Real Life Data from a Single Center Register

BackgroundConventional induction therapy of acute myeloid leukemia (AML) is still largely based on the combination of cytarabine (ARA-C) and daunorubicin. In the last two decades alternative drug combinations have been tested in order to improve complete remission (CR) rate and quality of remission. Fludarabine has been shown to enhance ara-CTP accumulation in leukemic blasts and to inhibit DNA repair mechanisms, thus providing a rationale for combination with DNA damaging agents. In our institution a fludarabine containing induction regimen (FLAI-5) is being used since more than 15 years.Patients and methodsEighty-four consecutive non M3 AML patients (age 17-72 years) treated in our center between 2006 and 2013 were retrospectively analyzed. Median follow-up was 42 months.Induction regimen included fludarabine 30 mg/sqm and ARA-C 2g/sqm on days 1 to 5 plus idarubicin 10 mg/sqm on days 1-3-5, with or without gemtuzumab ozogamicin (3mg/sqm) on day 6.Patients achieving complete remission received a second course including ARA-C 2g/sqm on days 1 to 5 and IDA at an increased dose of 12 mg/sqm on days 1-3-5.Patients were stratified in prognostic risk groups according to a comprehensive score based on karyotype, de-novo or secondary disease, NPM and FLT3 status. High-risk and selected intermediate-risk patients underwent allogeneic bone marrow transplantation (BMT) in first complete remission if a donor was available. The other patients were scheduled to receive at least 2 and up to 4 courses of consolidation therapy with 4 days of ARA-C (2g/sqm, daily). Patients features, CR rate and overall survival (OS)analysis are summarized in Table 1.Results and DiscussionSix patients (7%) died during first induction mainly because of infective or hemorrhagic events. Of the remaining 78 patients, 63 patients achieved CR (81%) and 15 did not respond (19%). FLAI-5 was generally well tolerated, with negligible non-hematological toxicity; median time to neutrophil and platelet recovery was 18 and 17 days, respectively. Most patients were able to receive subsequent therapy at full dose and in a timely manner; 33/63 CR patients underwent BMT whereas 21/30 (70%) non-transplanted patients received the full programmed therapy. In the whole cohort, 3 years OS was 50.9% (median 38 months). Factors affecting CR rates and OS are summarized in Tab.1.In a large randomized trial Burnett et al showed that FLAG-Ida favorably compared with other induction schedule. However the longer disease free survival and the lower relapse rate observed in the FLAG-Ida arm did not translate in a higher OS mostly due to the severe hematological toxicities following the second course.Our results are comparable to those recently published and show that the administration of fludarabine only in the first course may significantly reduce hematological toxicity therefore allowing patients to proceed along the consolidation program or undergo allo-BMT in a good performance status. Besides, we were able to increase IDA dose (10 and 12mg/sqm in first and second course, respectively, compared to 8 mg/sqm used in the FLAG-Ida combination).Our results show that FLAI-5 induction regimen followed by a second ARA-C and IDA containing course is an effective and well tolerated therapy for younger AML patients and confirm that CR rate and OS are mainly affected by comprehensive risk group, WBC count at diagnosis and NPM status.Abstract 2266. Table 1:Patients features and CR/OS analysisCR after 1st cycle/Tot.(%)p(univ.)p(multiv.)Median OS(months)OS 36 months (%)p(univ.)P(multiv.)All Patients63/78 (81)--3850.9--Age< 45 yrs28/34 (82)1.000-NR53.40.1210.105> 45 yrs35/44 (80)1549.2KaryotypeFavorable3/3 (100)0.0930.248NR1000.0030.775Intermediate52/61 (85)NR60.9Sfavorable8/13 (62)70NPMWild type36/50 (72)0.0030.0013245.10.2700.541Mutated26/26 (100)6566FLT3Wild type48/60 (80)0.721-6556.30.2490.658FLT3 ITD14/16 (88)1435Disease OnsetDe novo57/67 (85)0.0310.2136556.10.0010.809Secondary6/11 (55)721.4WBC at diagnosis<30.000/mmc41/47 (87)0.0870.0206559.40.0920.326>30.000/mmc22/31 (71)1439.4ComprehensiveRisk GroupGood17/17 (100)0.0090.983NR81.20.0000.001Intermediate31/37 (84)NR63.5Poor15/24 (63)914.4MylotargFLAI44/54 (82)1.000-3449.10.676-MY-FLAi19/24 (79)6552 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacokinetics of Fludarabine in Pediatric Hematopoietic Stem Cell Transplantation

▪Introduction: Fludarabine is a purine-analog which is effective for leukemic cells with lower toxicity. Thus it has been preferred for preparative regimens of hematopoietic stem cell transplantation (HSCT) recently. However, the pharmacokinetics of fludarabine in pediatric HSCT has not been studied before. This prospective study investigated the pharmacokinetics of fludarabine in children undergoing allogeneic HSCT, and attempted to establish the fludarabine administration in pediatric patients.Patients and Methods: Forty-three pediatric patients undergoing HSCT were enrolled to the study. The median age was 11.8 years old (range 1.3–17.3), and there were 31 male and 12 female patients. Among the 43 patients, there were 15 acute lymphoblastic leukemia (34.9%), 12 acute myeloid leukemia (27.9%), 3 severe aplastic anemia (7.0%), 3 chronic granulomatous disease (7.0%), and 10 other diseases (23.3%). The preparative regimens included cyclophosphamide with fludarabine, busulfan with fludarabine, busulfan with fludarabine and etoposide, which was selected according to the disease and risk group. Fludarabine was administered as 40 mg/m2/day i.v. over 30 minutes for 5 to 6 days, and the pharmacokinetic study was carried out at the first and last dose. Blood samplings were taken before administration and 0.5, 1, 3, 5, 8hr after the end of infusion. Fludarabine concentration was analyzed by high performance liquid chromatography-tandem mass spectrometry.Results: Median (min-max) fludarabine area under the drug concentration-time curve extrapolated to infinity (AUC0-∞) of the first day of infusion was 4.64 (2.71-9.52) μg*h/mL, apparent clearance 10.9 (3.28-26.49) L/h, and Cmax 1,222 (668-1,732) ng/mL. The AUC0-∞ was lower than previously reported AUC0-∞ of the adult study, but the median Cmax was higher than the result of adult study. In this study, the range of AUC and Cmax were narrower than those of adult data. When the AUC0-8hr of day 1 and the steady state (day 5 or day 6) was compared, the fludarabine exposure at steady state was 1.21 fold higher than the first day.In this study, the overall survival was 75.8%, and event-free survival was 60.9%. When grouped by median fludarabine AUC level, the high AUC group and low AUC group showed no significant difference in overall survival or relapse-free survival. Also, there was no significant difference in cumulative incidence of relapse or treatment-related mortality (TRM) between two groups. Neurotoxicity was observed in 5 patients (11.6%) and pulmonary toxicity was observed in 19 patients (44%). These toxicities were not significantly related to the level of fludarabine AUC.Conclusion: In this study, the fludarabine AUC(0-∞) and Cmax was similar with adults, and the range was narrower. Thus fludarabine exposure is considered to be similar with adult, and the recommended dosing for adults can be applied to children. This is the first study to investigate the pharmacokinetics of fludarabine in pediatric HSCT. As fludarabine is being more widely adopted for the pediatric HSCT, this study could provide useful data for the treatment in pediatric patients.Acknowledgment: This research was supported by a grant (11172MFDS288) from Ministry of Food and Drug safety in 2011. DisclosuresNo relevant conflicts of interest to declare.