Hiroto Inaba

NKAML: A Pilot Study to Determine the Safety and Feasibility of Haploidentical Natural Killer Cell Transplantation in Childhood Acute Myeloid Leukemia

PURPOSE To conduct a pilot study to determine the safety, feasibility, and engraftment of haploidentical natural killer (NK) cell infusions after an immunosuppressive regimen in children with acute myeloid leukemia (AML). PATIENTS AND METHODS Ten patients (0.7 to 21 years old) who had completed chemotherapy and were in first complete remission of AML were enrolled on the Pilot Study of Haploidentical Natural Killer Cell Transplantation for Acute Myeloid Leukemia (NKAML) study. They received cyclophosphamide (60 mg/kg on day -7) and fludarabine (25 mg/m(2)/d on days -6 through -2), followed by killer immunoglobulin-like receptor-human leukocyte antigen (KIR-HLA) mismatched NK cells (median, 29 x 10(6)/kg NK cells) and six doses of interleukin-2 (1 million U/m(2)). NK cell chimerism, phenotyping, and functional assays were performed on days 2, 7, 14, 21, and 28 after transplantation. Results All patients had transient engraftment for a median of 10 days (range, 2 to 189 days) and a significant expansion of KIR-mismatched NK cells (median, 5,800/mL of blood on day 14). Nonhematologic toxicity was limited, with no graft-versus-host disease. Median length of hospitalization was 2 days. With a median follow-up time of 964 days (range, 569 to 1,162 days), all patients remain in remission. The 2-year event-free survival estimate was 100% (95% CI, 63.1% to 100%). CONCLUSION Low-dose immunosuppression followed by donor-recipient inhibitory KIR-HLA mismatched NK cells is well tolerated by patients and results in successful engraftment. We propose to further investigate the efficacy of KIR-mismatched NK cells in a phase II trial as consolidation therapy to decrease relapse without increasing mortality in children with AML.

Minimal residual disease-directed therapy for childhood acute myeloid leukaemia: results of the AML02 multicentre trial

BACKGROUND We sought to improve outcome in patients with childhood acute myeloid leukaemia (AML) by applying risk-directed therapy that was based on genetic abnormalities of the leukaemic cells and measurements of minimal residual disease (MRD) done by flow cytometry during treatment. METHODS From Oct 13, 2002, to June 19, 2008, 232 patients with de-novo AML (n=206), therapy-related or myelodysplasia-related AML (n=12), or mixed-lineage leukaemia (n=14) were enrolled at eight centres. 230 patients were assigned by block, non-blinded randomisation, stratified by cytogenetic or morphological subtype, to high-dose (18 g/m(2), n=113) or low-dose (2 g/m(2), n=117) cytarabine given with daunorubicin and etoposide (ADE; induction 1). The primary aim of the study was to compare the incidence of MRD positivity of the high-dose group and the low-dose group at day 22 of induction 1. Induction 2 consisted of ADE with or without gemtuzumab ozogamicin (GO anti-CD33 monoclonal antibody); consolidation therapy included three additional courses of chemotherapy or haematopoietic stem-cell transplantation (HSCT). Levels of MRD were used to allocate GO and to determine the timing of induction 2. Both MRD and genetic abnormalities at diagnosis were used to determine the final risk classification. Low-risk patients (n=68) received five courses of chemotherapy, whereas high-risk patients (n=79), and standard-risk patients (n=69) with matched sibling donors, were eligible for HSCT (done for 48 high-risk and eight standard-risk patients). All 230 randomised patients were analysed for the primary endpoint. Other analyses were limited to the 216 patients with AML, excluding those with mixed-lineage leukaemia. This trial is closed to accrual and is registered with ClinicalTrials.gov, number NCT00136084. FINDINGS Complete remission was achieved in 80% (173 of 216 patients) after induction 1 and 94% (203 of 216) after induction 2. Induction failures included two deaths from toxic effects and ten cases of resistant leukaemia. The introduction of high-dose versus low-dose cytarabine did not significantly lower the rate of MRD-positivity after induction 1 (34%vs 42%, p=0.17). The 6-month cumulative incidence of grade 3 or higher infection was 79.3% (SE 4.0) for patients in the high-dose group and 75.5% (4.2) for the low-dose group. 3-year event-free survival and overall survival were 63.0% (SE 4.1) and 71.1% (3.8), respectively. 80% (155 of 193) of patients achieved MRD of less than 0.1% after induction 2, and the cumulative incidence of relapse for this group was 17% (SE 3). MRD of 1% or higher after induction 1 was the only significant independent adverse prognostic factor for both event-free (hazard ratio 2.41, 95% CI 1.36-4.26; p=0.003) and overall survival (2.11, 1.09-4.11; p=0.028). INTERPRETATION Our findings suggest that the use of targeted chemotherapy and HSCT, in the context of a comprehensive risk-stratification strategy based on genetic features and MRD findings, can improve outcome in patients with childhood AML. FUNDING National Institutes of Health and American Lebanese Syrian Associated Charities (ALSAC).

Glucocorticoid use in acute lymphoblastic leukaemia.

Glucocorticoids (prednisone and dexamethasone) play an essential part in the treatment of acute lymphoblastic leukaemia (ALL), but their optimum doses and bioequivalence have not been established. Results of preclinical studies have shown that dexamethasone has a longer half-life and better CNS penetration than does prednisone. In prospective randomised trials, dexamethasone improved control of CNS leukaemia. At a prednisone-to-dexamethasone dose ratio of less than seven, dexamethasone (6-18 mg/m(2) per day) resulted in a better event-free survival than did prednisone (40-120 mg/m(2) per day), and high-dose dexamethasone (10-18 mg/m(2) per day) improved the outcome of T-cell ALL and high-risk ALL. However, dexamethasone caused more adverse effects, including infection, bone fracture, osteonecrosis, mood and behaviour problems, and myopathy. At a dose ratio greater than seven, the two drugs showed no difference in efficacy. Therefore, the efficacy of prednisone and dexamethasone is dose dependent and needs to be carefully assessed against the toxic effects. Moreover, although dexamethasone generally showed increased activity in ALL cells in vitro, the dose ratio of the two drugs that exerted equivalent cytotoxic effects differed substantially in samples from individuals. The selection of the type and dose of glucocorticoid should be based on the risk of relapse, treatment phase, and the chemotherapeutic drugs used concomitantly.

Detectable minimal residual disease before hematopoietic cell transplantation is prognostic but does not preclude cure for children with very-high-risk leukemia.

In patients with acute leukemia, detection of minimal residual disease (MRD) before allogeneic hematopoietic cell transplantation (HCT) correlates with risk of relapse. However, the level of MRD that is most likely to preclude cure by HCT is unclear, and the benefit of further chemotherapy to reduce MRD before HCT is unknown. In 122 children with very-high-risk acute lymphoblastic leukemia (ALL; n = 64) or acute myeloid leukemia (AML, n = 58), higher MRD levels at the time of HCT predicted a poorer survival after HCT (P = .0019); MRD was an independent prognostic factor in a multivariate analysis (P = .0035). However, the increase in risk of death associated with a similar increment of MRD was greater in ALL than in AML, suggesting that a pretransplantation reduction of leukemia burden would have a higher impact in ALL. At any given MRD level, survival rates were higher for patients treated in recent protocols: the 5-year overall survival for patients with ALL was 49% if MRD was detectable and 88% if it was not and the corresponding rates for patients with AML were 67% and 80%, respectively. Although MRD before HCT is a strong prognostic factor, its impact has diminished and should not be regarded as a contraindication for HCT.

ReviewGlucocorticoid use in acute lymphoblastic leukaemia

Glucocorticoids (prednisone and dexamethasone) play an essential part in the treatment of acute lymphoblastic leukaemia (ALL), but their optimum doses and bioequivalence have not been established. Results of preclinical studies have shown that dexamethasone has a longer half-life and better CNS penetration than does prednisone. In prospective randomised trials, dexamethasone improved control of CNS leukaemia. At a prednisone-to-dexamethasone dose ratio of less than seven, dexamethasone (6-18 mg/m(2) per day) resulted in a better event-free survival than did prednisone (40-120 mg/m(2) per day), and high-dose dexamethasone (10-18 mg/m(2) per day) improved the outcome of T-cell ALL and high-risk ALL. However, dexamethasone caused more adverse effects, including infection, bone fracture, osteonecrosis, mood and behaviour problems, and myopathy. At a dose ratio greater than seven, the two drugs showed no difference in efficacy. Therefore, the efficacy of prednisone and dexamethasone is dose dependent and needs to be carefully assessed against the toxic effects. Moreover, although dexamethasone generally showed increased activity in ALL cells in vitro, the dose ratio of the two drugs that exerted equivalent cytotoxic effects differed substantially in samples from individuals. The selection of the type and dose of glucocorticoid should be based on the risk of relapse, treatment phase, and the chemotherapeutic drugs used concomitantly.

Comparative Analysis of Different Approaches to Measure Treatment Response in Acute Myeloid Leukemia

PURPOSE In acute myeloid leukemia (AML), initial treatment response by morphologic analysis of bone marrow predicts long-term outcome. Response can now be assessed by minimal residual disease (MRD) monitoring with flow cytometry or polymerase chain reaction (PCR). We determined the relation among the results of these approaches and their prognostic value. PATIENTS AND METHODS In the multicenter AML02 study, follow-up bone marrow samples from 203 children and adolescents with newly diagnosed AML were examined by flow cytometry (n = 1,514), morphology (n = 1,382), and PCR amplification of fusion transcripts (n = 508). Results were correlated with treatment outcome. RESULTS Among 1,215 samples with less than 5% leukemic myeloblasts by morphology, 100 (8.2%) were MRD positive (≥ 0.1%) by flow cytometry, whereas 96 (57.5%) of the 167 samples with ≥ 5% blasts were MRD negative. Virtually all (308 of 311; 99.0%) MRD-negative samples by PCR were also MRD negative by flow cytometry. However, only 19 (9.6%) of the 197 PCR-positive samples were flow cytometry positive, with analyses of AML1-ETO and CBFβ-MYH11 accounting for most discrepancies, whereas eight of 13 MLL-positive samples had detectable MRD by flow cytometry. MRD by flow cytometry after induction 1 or 2 predicted lower event-free survival and higher relapse rate (P < .001) and was an independent prognostic factor in a multivariable analysis; prediction was not improved by morphologic information or molecular findings. CONCLUSION In childhood AML, morphologic assessment of treatment response has limited value if MRD is measured by flow cytometry. MLL fusion transcripts can provide prognostic information in some patients, whereas monitoring of AML1-ETO and CBFβ-MYH11 transcripts is largely uninformative.

Phase I Pharmacokinetic and Pharmacodynamic Study of the Multikinase Inhibitor Sorafenib in Combination With Clofarabine and Cytarabine in Pediatric Relapsed/Refractory Leukemia

PURPOSE To assess the toxicity, pharmacokinetics, and pharmacodynamics of multikinase inhibitor sorafenib in combination with clofarabine and cytarabine in children with relapsed/refractory leukemia. PATIENTS AND METHODS Twelve patients with acute leukemia (11 with acute myeloid leukemia [AML]) received sorafenib on days 1 to 7 and then concurrently with cytarabine (1 g/m(2)) and clofarabine (stratum one: 40 mg/m(2), n = 10; stratum two [recent transplantation or fungal infection]: 20 mg/m(2), n = 2) on days 8 to 12. Sorafenib was continued until day 28 if tolerated. Two sorafenib dose levels (200 mg/m(2) and 150 mg/m(2) twice daily) were planned. Sorafenib pharmacokinetic and pharmacodynamic studies were performed on days 7 and 8. RESULTS At sorafenib 200 mg/m(2), two of four patients in stratum one and one of two patients in stratum two had grade 3 hand-foot skin reaction and/or rash as dose-limiting toxicities (DLTs). No DLTs were observed in six patients in stratum one at sorafenib 150 mg/m(2). Sorafenib inhibited the phosphorylation of AKT, S6 ribosomal protein, and 4E-BP1 in leukemia cells. The rate of sorafenib conversion to its metabolite sorafenib N-oxide was high (mean, 33%; range, 17% to 69%). In vitro, the N-oxide potently inhibited FLT3-internal tandem duplication (ITD; binding constant, 70 nmol/L) and the viability of five AML cell lines. On day 8, sorafenib decreased blast percentages in 10 of 12 patients (median, 66%; range, 9% to 95%). After combination chemotherapy, six patients (three FLT3-ITD and three FLT3 wild-type AML) achieved complete remission, two (both FLT3-ITD AML) had complete remission with incomplete blood count recovery, and one (FLT3 wild-type AML) had partial remission. CONCLUSION Sorafenib in combination with clofarabine and cytarabine is tolerable and shows activity in relapsed/refractory pediatric AML.

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

Widely used as anticancer and immunosuppressive agents, thiopurines have narrow therapeutic indices owing to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and the clinical implications of this pharmacogenetic association remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore and Japan, we identified four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile and p.Val18_Val19insGlyVal) that resulted in 74.4–100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across the three cohorts (P = 0.021, 2.1 × 10−5 and 0.0054, respectively; meta-analysis P = 4.45 × 10−8, allelic effect size = −11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased thiopurine cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive levels of thiopurine active metabolites and toxicity. Taken together, these results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy.

Crenolanib is active against models of drug-resistant FLT3-ITD-positive acute myeloid leukemia

FLT3 kinase internal tandem duplication (ITD) mutations are common in acute myeloid leukemia (AML) and are associated with poor clinical outcomes. Although initial responses to FLT3 tyrosine kinase inhibitors (TKIs) are observed in FLT3-ITD-positive patients, subsequent relapse often occurs upon acquisition of secondary FLT3 kinase domain (KD) mutations, primarily at residues D835 and F691. Using biochemical assays, we determined that crenolanib, a novel TKI, demonstrates type I properties and is active against FLT3 containing ITD and/or D835- or F691-activating mutations. Potent activity was observed in FLT3-ITD-positive AML cell lines. Crenolanib delayed the outgrowth of MV4-11 cells in a xenograft mouse model, whereas in combination with the type II TKI sorafenib, a significant decrease in leukemic burden (P < .001) and prolonged survival (P < .01) was observed compared with either type I or II TKI alone. Crenolanib was active against Ba/F3 cells harboring FLT3-ITD and secondary KD mutations and sorafenib-resistant MOLM-13 cells containing FLT3-ITD/D835Y both in vitro and in vivo. In addition, crenolanib inhibited drug-resistant AML primary blasts with FLT3-ITD and D835H/Y mutations. These preclinical data demonstrate that crenolanib is effective against FLT3-ITD containing secondary KD mutations, suggesting that crenolanib may be a useful therapeutic agent for TKI-naive and drug-resistant FLT3-ITD-positive AML.

Methotrexate-Induced Neurotoxicity and Leukoencephalopathy in Childhood Acute Lymphoblastic Leukemia

PURPOSE Methotrexate (MTX) can cause significant clinical neurotoxicity and asymptomatic leukoencephalopathy. We sought to identify clinical, pharmacokinetic, and genetic risk factors for these MTX-related toxicities during childhood acute lymphoblastic leukemia (ALL) therapy and provide data on safety of intrathecal and high-dose MTX rechallenge in patients with neurotoxicity. PATIENTS AND METHODS Prospective brain magnetic resonance imaging was performed at four time points for 369 children with ALL treated in a contemporary study that included five courses of high-dose MTX and 13 to 25 doses of triple intrathecal therapy. Logistic regression modeling was used to evaluate clinical and pharmacokinetic factors, and a genome-wide association study (GWAS) was performed to identify germline polymorphisms for their association with neurotoxicities. RESULTS Fourteen patients (3.8%) developed MTX-related clinical neurotoxicity. Of 13 patients rechallenged with intrathecal and/or high-dose MTX, 12 did not experience recurrence of neurotoxicity. Leukoencephalopathy was found in 73 (20.6%) of 355 asymptomatic patients and in all symptomatic patients and persisted in 74% of asymptomatic and 58% of symptomatic patients at the end of therapy. A high 42-hour plasma MTX to leucovorin ratio (measure of MTX exposure) was associated with increased risk of leukoencephalopathy in multivariable analysis (P = .038). GWAS revealed polymorphisms in genes enriched for neurodevelopmental pathways with plausible mechanistic roles in neurotoxicity. CONCLUSION MTX-related clinical neurotoxicity is transient, and most patients can receive subsequent MTX without recurrence of acute or subacute symptoms. All symptomatic patients and one in five asymptomatic patients develop leukoencephalopathy that can persist until the end of therapy. Polymorphisms in genes related to neurogenesis may contribute to susceptibility to MTX-related neurotoxicity.