Role of metabolites of cyclophosphamide in cardiotoxicity.

BACKGROUND: The dose-limiting toxic effect of cyclophosphamide (CY) is cardiotoxicity. The pathogenesis of myocardial damage is poorly understood, and there is no established means of prevention. In previous studies, we suggested that for CY-induced cardiotoxicity, whereas acrolein is the key toxic metabolite, carboxyethylphosphoramide mustard (CEPM) is protective. We sought to verify that acrolein is the main cause of cardiotoxicity and to investigate whether aldehyde dehydrogenase (ALDH), which is associated with greater CEPM production, is involved in the protective effect for cardiotoxicity. We also evaluated the protective effect of N-acetylcysteine (NAC), an amino acid with antioxidant activity and a known acrolein scavenger. METHODS: H9c2 cells were exposed to CY metabolites HCY (4-hydroxy-cyclophosphamide), acrolein or CEPM. The degree of cytotoxicity was evaluated by MTT assay, lactate dehydrogenase (LDH) release, and the production of reactive oxygen species (ROS). We also investigated how the myocardial cellular protective effects of CY metabolites were modified by NAC. To quantify acrolein levels, we measured the culture supernatants using high performance liquid chromatography. We measured ALDH activity after exposure to HCY or acrolein and the same with pre-treatment with NAC. RESULTS: Exposure of H9c2 cells to CEPM did not cause cytotoxicity. Increased ROS levels and myocardial cytotoxicity, however, were induced by HCY and acrolein. In cell cultures, HCY was metabolized to acrolein. Less ALDH activity was observed after exposure to HCY or acrolein. Treatment with NAC reduced acrolein concentrations. CONCLUSIONS: Increased ROS generation and decreased ALDH activity confirmed that CY metabolites HCY and acrolein are strongly implicated in cardiotoxicity. By inhibiting ROS generation, increasing ALDH activity and decreasing the presence of acrolein, NAC has the potential to prevent CY-induced cardiotoxicity.

Gene expression ratio as a predictive determinant of nelarabine chemosensitivity in T-lymphoblastic leukemia/lymphoma.

BACKGROUND: Nelarabine has been used for the treatment of T-cell malignancies including T-acute lymphoblastic leukemia (T-ALL)/T-lymphoblastic lymphoma. However, the mechanisms that underlie the susceptibility or resistance to nelarabine have not been fully elucidated. The aim of this study was to determine the significance of nelarabine transport and metabolism in the context of nelarabine cytotoxicity. PROCEDURE: The expression profiles of six genes in the nelarabine pathway were analyzed in blast cells from six patients with T-ALL as well as in three T-ALL cell lines. In vitro cytotoxicity (LC50 of 9-ß-d-arabinofuranosylguanine [ara-G]) was evaluated. RESULTS: The mRNA expression of ENT1, DCK, CDA, NT5C2, RRM1, and RRM2 in patients showed inter-individual variability and was not correlated with the LC50 of ara-G. However, the ratio of (ENT1 × DCK)/(CDA × RRM1) expression was significantly correlated with LC50 (r = -0.831, P = 0.0405). CONCLUSIONS: Chemosensitivity to nelarabine is influenced by the balance of the expression of these four genes, and the ratio of their expression predicts the response of T-cell malignancies to nelarabine.

Central venous catheter-related blood stream infection with pyomyositis due to Stenotrophomonas maltophilia after allogeneic bone marrow transplantation in a patient with aplastic anemia.

Stenotrophomonas maltophilia causes pneumonia and CVC-CRBSI in HSCT. However, there are few reports of pyomyositis due to S. maltophilia. We report a patient with CRBSI and pyomyositis due to S. maltophilia after allogeneic HSCT who was successfully treated by removing the CVC and antibiotics without surgical drainage. Removing the CVC and the combined antibiotics without preventing the neutrophil engraftment could avoid surgical drainage in pyomyositis due to S. maltophilia when detected in an early stage.

Effect of Cytomegalovirus Reactivation on Relapse after Allogeneic Hematopoietic Stem Cell Transplantation in Pediatric Acute Leukemia.

Recent studies have demonstrated the protective effect of cytomegalovirus (CMV) reactivation against relapse after allogeneic hematopoietic stem cell transplantation (HSCT) for adult myeloid malignancies. We assessed the association of CMV reactivation, defined as the development of CMV antigenemia (at least 1 pp65 antigen-positive cell per 5.0 × 10(4) WBCs) within 100 days after HSCT, with the risk of relapse in 143 patients with pediatric acute leukemia. The median age at HSCT was 7 years, and underlying diseases included acute lymphoblastic leukemia in 101 patients and acute myeloid leukemia in 42. The cumulative incidence of CMV reactivation at day 100 after HSCT was 45.4%. At a median follow-up of 88 months, patients with CMV reactivation had significantly lower 5-year cumulative incidence of relapse compared with patients without CMV reactivation. In a multivariate analysis, high-level CMV reactivation (≥10 pp65 antigen-positive cells) was an independent factor associated with reduced relapse. However, CMV reactivation was also associated with higher nonrelapse mortality (NRM), mostly caused by opportunistic infection after grades II to IV acute graft-versus-host disease (GVHD), which resulted in decreased probability of survival. High-level CMV reactivation was a risk factor for increased NRM and worse overall survival in multivariate analysis. Although CMV reactivation may reduce the risk of relapse after HSCT for pediatric acute leukemia, effective management of severe acute GVHD and better prophylaxis and treatment of opportunistic infections are required to reduce the incidence of NRM and improve survival. Further studies on pediatric HSCT that include a larger number of patients and more homogenous patient cohorts are desirable.

Trough level monitoring of intravenous busulfan to estimate the area under the plasma drug concentration-time curve in pediatric hematopoietic stem cell transplant recipients.

Optimizing systemic busulfan exposure, the area under the concentration-time curve (AUC), improves the outcomes for hematopoietic stem cell transplantation (HSCT). The AUC is conventionally calculated using six plasma concentrations (AUC(0-∞)) drawn after the first of 16 intravenous busulfan doses given as a 2-h infusion every 6 h. The aim of the present study was to develop limited sampling strategies using three or fewer busulfan concentrations to reliably calculate AUC(0-∞) in patients undergoing HSCT. We investigated the pharmacokinetics of busulfan 46 times in 29 pediatric patients receiving intravenous busulfan. Limited sampling strategies using one, two, or three plasma busulfan concentrations were developed by multiple linear regression that showed excellent agreement with AUC(0-∞). In single-point sampling strategies, the AUC(0-∞) predicted based on C(6) (trough level: busulfan plasma concentration 6 h after the start of the infusion) was significantly correlated with, and not statistically different from, actual values as follows: AUC(0-∞) = 2556.5 C6 + 320.9 (r(2) = 0.929, P < 0.0001, mean bias 0.282 %, precision 7.91 %). In contrast, the predicted AUCs derived from the other sampling single points did not meet the criteria. The trough level well correlated with actual AUC(0-∞), suggesting that this time-point is acceptable for busulfan monitoring.

Long-Term Morbidity and Mortality in Children with Chronic Graft-versus-Host Disease Classified by National Institutes of Health Consensus Criteria after Allogeneic Hematopoietic Stem Cell Transplantation.

We report the long-term morbidity and mortality of 105 pediatric patients who developed chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). According to the consensus criteria of the National Institutes of Health, the global severity of cGVHD was mild in 26 patients (25%), moderate in 30 patients (29%), and severe in 49 patients (47%). Patients with severe cGVHD had a significantly lower cumulative incidence of cGVHD remission and higher probability of continuing cGVHD at 8 years from cGVHD diagnosis compared with those with mild or moderate cGVHD. The 10-year cumulative incidence of nonrelapse mortality in severe cGVHD patients was significantly higher and the probability of disease-free survival was significantly lower than those among patients with mild and moderate cGVHD. Of the 59 patients who survived for more than 5 years, 20 (34%) (4 with moderate and 16 with severe cGVHD) had persistent functional impairment caused by cGVHD with a Karnofsky/Lansky performance score of 90% in 3 patients, 80% in 4 patients, and below 70% in 13 patients at the time of relapse, death, or last follow-up. Better therapeutic strategies are needed to lower the incidence of severe cGVHD, considering the longer life expectancy of pediatric HSCT survivors.

Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention.

Observed only after administration of high doses, cardiotoxicity is the dose-limiting effect of cyclophosphamide (CY). We investigated the poorly understood cardiotoxic mechanisms of high-dose CY. A rat cardiac myocardial cell line, H9c2, was exposed to CY metabolized by S9 fraction of rat liver homogenate mixed with co-factors (CYS9). Cytotoxicity was then evaluated by 3-(4,5-dimethyl-2-thiazolyl)¬2,5-diphenyl¬2H-tetrazolium bromide (MTT) assay, lactate dehydrogenase release, production of reactive oxygen species (ROS), and incidence of apoptosis. We also investigated how the myocardial cellular effects of CYS9 were modified by acrolein scavenger N-acetylcysteine (NAC), antioxidant isorhamnetin (ISO), and CYP inhibitor ß-ionone (BIO). Quantifying CY and CY metabolites by means of liquid chromatography coupled with electrospray tandem mass spectrometry, we assayed culture supernatants of CYS9 with and without candidate cardioprotectant agents. Assay results for MTT showed that treatment with CY (125-500 µM) did not induce cytotoxicity. CYS9, however, exhibited myocardial cytotoxicity when CY concentration was 250 µM or more. After 250 µM of CY was metabolized in S9 mix for 2 h, the concentration of CY was 73.6 ± 8.0 µM, 4-hydroxy-cyclophosphamide (HCY) 17.6 ± 4.3, o-carboxyethyl-phosphoramide (CEPM) 26.6 ± 5.3 µM, and acrolein 26.7 ± 2.5 µM. Inhibition of CYS9-induced cytotoxicity occurred with NAC, ISO, and BIO. When treated with ISO or BIO, metabolism of CY was significantly inhibited. Pre-treatment with NAC, however, did not inhibit the metabolism of CY: compared to control samples, we observed no difference in HCY, a significant increase of CEPM, and a significant decrease of acrolein. Furthermore, NAC pre-treatment did not affect intracellular amounts of ROS produced by CYS9. Since acrolein seems to be heavily implicated in the onset of cardiotoxicity, any competitive metabolic processing of CY that reduces its transformation to acrolein is likely to be an important mechanism for preventing cardiotoxicity.

Hematopoietic stem cell transplantation following unsuccessful salvage treatment for relapsed acute lymphoblastic leukemia in children.

BACKGROUND: For children who experience a re-induction failure or multiple recurrences following the first relapse of acute lymphoblastic leukemia (ALL), it is uncertain whether additional intensive chemotherapy aimed at hematopoietic stem cell transplantation (SCT) in complete remission (CR) or immediate SCT even in non-CR should be performed. This study aimed to investigate the impact of disease status at SCT on the outcomes of SCT for these children, whose prognosis is considered unquestionably poor even with SCT. PROCEDURE: The medical records of 55 children with ALL who underwent SCT following the experience of re-induction failure (n = 25) or multiple relapses (n = 30) were retrospectively analyzed. RESULTS: Twenty-one patients underwent SCT in CR (delayed CR2, CR3, and CR4) and 34 in non-CR (first or subsequent relapse). The probability of overall survival of patients with CR and with non-CR at SCT was 42.9% and 23.5% (P = 0.15), leukemia-free survival was 38.1% and 20.6% (P = 0.18), and the cumulative incidence of relapse at 2 years was 23.8% and 50%, respectively (P = 0.05). In multivariate analysis, non-CR at SCT was a significant risk factor for higher relapse incidence and male sex was a significant risk factor for lower survival. CONCLUSIONS: Our results indicated that in case of tolerable patient condition, further re-induction chemotherapy might be reasonable so that SCT could be performed in CR, which might result in a low incidence of relapse after SCT. Novel approaches are required to induce CR for the treatment of children with relapsed/refractory ALL.

Hematopoietic stem cell transplantation in children with refractory cytopenia of childhood: single-center experience using high-dose cytarabine containing myeloablative and aplastic anemia oriented reduced-intensity conditioning regimens.

Refractory cytopenia of childhood (RCC) is the most common subtype of myelodysplastic syndrome in children, and the clinical course of RCC is heterogeneous. A certain proportion of RCC patients need allogeneic hematopoietic stem cell transplantation (HSCT); however, data on HSCT outcomes are not abundant, and the optimal intensity of a preparative conditioning regimen remains uncertain. In this study, we evaluated the outcomes of HSCT in 24 patients with RCC. Eleven patients received myeloablative conditioning (MAC) consisting of high-dose cytarabine, cyclophosphamide, and either total body irradiation (TBI) or busulfan. Nine patients (38%) received a reduced-intensity conditioning (RIC) regimen; of these, 7 received low-dose TBI and cyclophosphamide (200 mg/kg) with or without antithymocyte globulin or fludarabine, and 2 patients received low-dose TBI, fludarabine, and melphalan (140 mg/m(2)). The remaining 4 patients had disease progression before HSCT and received the MAC regimen. With a median follow-up of 91 months (range, 6 to 263), the probability of overall survival at 5 years was 81.1% (95% CI, 57.0 to 92.5). The 5-year overall survival for the 15 patients who received MAC was 73.3% (95% CI, 43.6 to 89.1), and all 9 patients with RIC are alive without any events. Further study is needed to evaluate the efficacy of RIC for children with RCC with an expectation for reduction of late effects such as growth retardation and infertility.

Acute encephalomyelitis complicated with severe neurological sequelae after intrathecal administration of methotrexate in a patient with acute lymphoblastic leukemia.

A four-year-old girl on maintenance therapy for acute lymphoblastic leukemia (ALL) complained of a headache and low back pain on the day she received her 21st intrathecal methotrexate (it-MTX) administration, and the next day experienced numbness and pain in her foot. This numbness gradually spread to her hand. She thereafter developed a fever and was hospitalized on day 8. After antibiotic therapy, the fever disappeared. However, her lower limbs became paralyzed, and she also developed urinary retention. On day 12, her paralysis progressed upwards, and she also developed paralysis of the upper limbs. Finally, she experienced convulsions with an impairment of consciousness. A magnetic resonance imaging study of the brain and spinal cord showed abnormal signals in the brain cortex and anterior horn. Accordingly, we diagnosed acute encephalomyelitis associated with it-MTX. High-dose intravenous immunoglobulin, steroid pulse therapy, plasma exchange, and dextromethorphan administration were initiated, while she received mechanical ventilation. Despite this intensive treatment, she suffered severe neurological damage and had to be maintained on mechanical ventilation due to persistent flaccid quadriplegia one year after the onset. When patients have symptoms of ascending paralysis during it-MTX treatment, clinicians should carefully consider the possibility of acute encephalomyelitis due to it-MTX.

Bone marrow transplant for a girl with bone marrow failure and cerebral palsy.

Bone marrow transplantation (BMT) has been used with increasing frequency to treat congenital bone marrow failure syndrome (CBMFs) successfully. Decision to perform BMT, however, is difficult in the case of comorbidity because of regimen-related toxicities. We describe here a child with CBMFs, severe cerebral palsy (CP) at Gross Motor Function Classification System level V and mental retardation (MR) who was transfusion dependent despite various medications. She underwent BMT from an HLA-1 locus-mismatched unrelated donor. Although engraftment was successful, no neurological improvement was seen 5 years after BMT. While CBMFs patients who have CP and MR could undergo transplantation safely, they may not benefit neurologically from BMT.

Ramsay Hunt syndrome in a girl with acute lymphoblastic leukemia during maintenance therapy.

A 5-year-old girl with precursor B-cell acute lymphoblastic leukemia developed peripheral-type right facial palsy and very faint erythema on her right pinna during maintenance therapy. Acyclovir was started for possible zoster infection. The following day, vesicles appeared and a diagnosis of Ramsay Hunt syndrome was made. Prednisolone was started on day 5 after onset. Her facial palsy recovered within 6 months. Ramsay Hunt syndrome is a rare cause of facial palsy in patients with acute lymphoblastic leukemia, and this is the first case report. Preemptive therapy with acyclovir before the development of vesicles should help the patient recover from facial palsy.