Lene Molgaard-Hansen

Merja Möttönen

Heidi Glosli

Guðmundur K Jónmundsson

Jonas Abrahamsson

Henrik Hasle

Kurt Bønnelycke

Grethe Bønnelycke

Foundation The

M Brogaard

Foundation The

Anna Friedmann

Grant The

Simon Fougner Hartmann

Family Foundation The

Sophus Jacobsen

Astrid Jacobsen

Foundation The

Viggo Grove

Early and treatment-related deaths in childhood AML 1 Early and treatment-related deaths in childhood acute myeloid leukaemia in the Nordic countries: 1984-2003

Keywords: Acute myeloid leukaemia, children, early deaths, treatment-related mortality, infection

Early and treatment-related deaths in childhood AML 3

Introduction

The last two decades have seen a considerable improvement in the prognosis for children with acute myeloid leukaemia (AML) owing to the introduction of more intensive chemotherapy, better risk-group stratification and improved supportive care.

The overall survival rate in childhood AML has reached 60-65% [START_REF] Kaspers | Pediatric acute myeloid leukemia: international progress and future directions[END_REF]. Most deaths are due to progressive disease, but 5-15% die from toxic effects of treatment [START_REF] Riley | Treatment-related deaths during induction and first remission of acute myeloid leukaemia in children treated on the Tenth Medical Research Council acute myeloid leukaemia trial (MRC AML10). The MCR Childhood Leukaemia Working Party[END_REF][START_REF] Creutzig | Early deaths and treatment-related mortality in children undergoing therapy for acute myeloid leukemia: analysis of the multicenter clinical trials AML-BFM 93 and AML-BFM 98[END_REF][START_REF] Rubnitz | Death during induction therapy and first remission of acute leukemia in childhood: the St. Jude experience[END_REF][START_REF] Slats | Causes of death--other than progressive leukemia--in childhood acute lymphoblastic (ALL) and myeloid leukemia (AML): the Dutch Childhood Oncology Group experience[END_REF]. Further improvement in AML survival rates will probably require introduction of individualized therapy where targeted and more leukaemia-specific drugs are used in an effort to arrest progressive disease in combination with better supportive care to prevent early deaths (ED) and treatment-related deaths (TRD).

The aim of the present study was to identify the frequency, the clinical features and possible risk factors for EDs and TRDs in childhood AML in the Nordic countries. AML-84/88/93 or pre-treated with cytostatic drugs for more than 14 days, patients with myeloid leukaemia of Down syndrome, Fanconi anaemia, Kostmann syndrome, extramedullary myeloid tumour without significant bone marrow involvement, preceding myelodysplastic syndrome or therapy-related AML. A total of 525 patients fulfilled the inclusion criteria. Among these 489 were younger than 15 years at the time of diagnosis and 36 were 15-18 years old. The patients were diagnosed in 21 hospitals in Denmark (n=114), Finland (n=97), Iceland (n=6), Norway (n=106) and Sweden (n=202). The allogeneic transplants were performed in eight centres.

The Nordic AML database contains information about the time and cause of death, and 10 children dying before starting treatment, and 78 dying from treatment-related complications were identified. Information about patients registered as dying from treatment-related complications in second or third complete remission is not reported. Additional data concerning the remaining 70 patients were collected by reviewing each patient's file using a specific registration form. The 1 and2. Details concerning patient characteristics and clinical outcome have been reported elsewhere [START_REF] Lie | Long-term results in children with AML: NOPHO-AML Study Group--report of three consecutive trials[END_REF].

The NOPHO-AML-84 protocol was of moderate intensity with an induction of three courses of cytarabine, 6-thioguanine and doxorubicin followed by four consolidation courses with high-dose cytarabine. To reduce the frequency of non-response and relapse in the NOPHO-AML-88 study, etoposide and mitoxantrone were added in selected courses during induction and consolidation, and the interval between induction courses was reduced. The event-free survival rose from 29% to 41%, but the NOPHO-AML-88 protocol suffered from an unacceptably high frequency of toxic deaths during induction and in early consolidation. The NOPHO-AML-93 used the same induction blocks as the NOPHO-AML-88, but the dose intensity was modified: children with a good response after the first course were given their second course only following haematological recovery. Consolidation was identical with NOPHO-AML-88. The event-free survival at five years was 48% in the NOPHO-AML-93 protocol.

Intrathecal methotrexate was given on the first day of all courses. In the NOPHO-AML-84/88/93 studies, allogeneic haematopoietic stem cell transplantation (HSCT) in first complete remission (CR1) was recommended to all patients with a HLA-matched family donor. For patients without a donor, autologous HSCT was optional in the NOPHO-AML-84, -88 and the first part of -93, and consequently, autologous HSCT was performed on a non-randomised basis decided by the responsible physician. HSCT conditioning regimens and supportive care followed local guidelines.

Recommendations for supportive care measures in the NOPHO-AML-84/88/93 protocols included trimethoprim/sulfamethoxazole prophylaxis for Pneumocystis jiroveci. Systemic antifungal prophylaxis and granulocyte-colony-stimulating factor (G-CSF) were not recommended. Cytoreduction prior to chemotherapy was performed at the discretion of the attending physician. All-trans-retinoic acid (ATRA) was recommended to patients with acute promyelocytic leukaemia (APL) as from 1999.

Definitions

The diagnosis of AML was established by morphological analysis of bone marrow (BM) aspirates according to the FAB and WHO classifications.

Central nervous system (CNS) involvement was defined as more than five leucocytes per microlitre in the cerebrospinal fluid (CSF) in combination with detectable leukaemic cells in the cytospin and/or presence of neurological symptoms such as cranial nerve palsy.

Complete remission (CR) was defined according to the CALGB criteria [START_REF] Cheson | Report of the National Cancer Institute-sponsored workshop on definitions of diagnosis and response in acute myeloid leukemia[END_REF] with minor deviations: <5% leukaemic blasts in the BM with signs of normal haematopoiesis in the BM and with clear signs of regeneration of normal blood cell production in the peripheral blood (PB) (platelets >80 x 10 9 /l without transfusions and neutrophils >1.0 x 10 9 /l), and no leukaemic cells in the PB or in the CSF. Response after induction was evaluated close to day 15; a blast cell count in the BM less than 5% was considered as good response.

Non-responders (NR) included all patients surviving the first 42 days of treatment without achieving CR. Patients with CR criteria lasting less than four weeks were also classified as NR. NRs at day 42 (n=29) were censored in the analysis of TRD.

Early death (ED) was defined as a fatal event occurring before or within the first 42 days of treatment. ED was subclassified into: a) ED 0-14 before or during and after the first therapy course (<15 days of treatment) and b) ED 15-42 in aplasia between days 15-42 from treatment start. This classification reflects the ED rate and its association with initial problems (hyperleukocytosis, leukostasis) or aplasia after induction therapy.

Treatment-related death (TRD) was defined as death unrelated to progressive disease occurring after day 42 of treatment start in patients who achieved complete remission. In this paper, only TRDs in CR1 are reported.

Hyperleukocytosis was defined as WBC >100 x 10 9 /l at diagnosis.

Infections were categorized as clinically, radiologically or microbiologically documented. Viral disease was registered when both relevant disease symptoms and virus were detected. Fungal infections were defined as proven invasive infection (positive histopathological findings or positive culture) [START_REF] Ascioglu | Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus[END_REF].

Cytogenetics: Patients with t(8;21)(q22;q22), t(9;11)(p22;q23), t(15;17)(q22;q12-21), inv16(p13q22) and t(16;16)(p13;q22) were classified as having a favourable karyotype [START_REF] Grimwade | The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties[END_REF]. Kaplan-Meier plots were used for survival analysis, including estimation of cumulative incidence of EDs and TRDs. The main event in the analysis of possible risk factors was composite endpoints consisting of ED 15-42 or TRD. Time was defined as time from day 15 after diagnosis to date of death. Patients who experienced a relapse (n=203) or a second malignant neoplasm (n=2) were censored at the time of this event. Patients without events were censored at the time of last follow-up. Cox proportional hazard regression analysis was performed with the composite endpoints as events and time to this as outcome measure. The following covariates were included: age at diagnosis (<2, 2-9, >10 years), WBC at diagnosis (<20, 20-99, >100

x 10 9 /l) and protocol (NOPHO-AML-84 vs. NOPHO-AML-88 or NOPHO-AML-93). For patients who underwent HSCT, a time-dependent covariate was defined as zero before, and one after the date of HSCT, and for all other patients, this timedependent covariate was defined as zero.

A stepwise, multiregression analysis according to Cox was also performed for composite endpoints consisting of ED 15-42 or TRD caused by infection. The analysis was adjusted for age at diagnosis and protocol. 3). ED and TRD rose from 11% in the NOPHO-AML-84 protocol to 29% in -88 and then declined to 8% in -93. The frequency of EDs and TRDs in the first half of the protocol period compared with the second half of the protocol was 9% vs. 14% in NOPHO-AML-84 (p=0.5), 35% vs. 24% in NOPHO-AML-88 (p=0.2), and 9% vs. 8% in NOPHO-AML-93 (p=0.8).

The analysis was performed in February 2010 with a median follow-up of 11.8 years (range, 2.8-25.1 years) for patients registered as alive.

Early death day 0-14

The main causes of ED 0-14 and characteristics of patients appear from Table 4. The death rate was largely unchanged over time with 3%, 6% and 2% in the NOPHO-AML-84, -88 and -93 protocols, respectively (p=0.4 and p=0.05 when comparing NOPHO-AML-93 with -84 and -88, respectively). Ten (63%) patients died before protocol treatment was begun and three shortly after the first induction course.

Among the 16 patients (3%) who died early, 11 (69%) died from intracranial haemorrhage, three (19%) from leukostasis and two (12%) from sepsis. Age was a risk factor for ED 0-14 : 87% (n=14) of the patients were <2 years or >10 years at diagnosis (p=0.02). Hyperleukocytosis was also a risk factor. It was present in 75% of the ED 0-14 patients compared with 15% of the AML cases in general (p<0.001). FAB M4 or M5 was not a significant risk factor for ED 0-14 compared with other FAB-types (p=0.2). However, 55% of the patients who died from a cerebral haemorrhage had FAB types M4 or M5, vs. 38% in the AML population (p=0.1).

Early death day 15-42

The principal cause of death among the 15 patients (3%), who died between days 15-42, was infection (93%) (Table 5). Four (29%) already had infection signs at the time of their last course of chemotherapy, which was the first induction course in one case. One patient died from a gastrointestinal bleeding after the second induction course.

Treatment-related death (> day 42)

TRD occurred in connection with chemotherapy in 26 (5%) patients. The main cause of death was infection (n=22, 85%) (Table 5). Eight (36%) of these patients had signs of infection (fever and/or respiratory symptoms) when the last course of chemotherapy before death was being administered. One patient with FAB M4 died from a cerebral bleeding. Two patients died from acute respiratory distress syndrome (ARDS). A 13-year-old girl died unexpectedly one day after the first consolidation course. She had been complaining of chest pain, but the autopsy revealed no signs of heart disease or other causes of death.

TRD occurred in ten (7%) of 150 patients transplanted in CR1. Eight (80%) of these deaths were caused by a severe infection. One patient died from veno-occlusive disease (VOD) and another from graft-versus-host-disease (GVHD) with bronchiolitis obliterans.

Three patients, all treated with chemotherapy only, died from TRD more than one year after end of AML treatment. A 13-year-old female died from cardiac failure 11 6). Eighteen (13%) of 141 patients <2 years of age (p=0.03) and 23 (14%) of 169 patients >10 years (p=0.01) died from treatment-related complications as compared with 13 (6%) of 215 children between 2 and 9 years of age. Another risk factor was treatment according to the NOPHO-AML-88 protocol (p=0.01). The number of ED 15-42 or TRDs was 26 (23%) in the NOPHO-AML-88 as compared with 8 (8%) and 20 (6%) in the NOPHO-AML-84 and -93, respectively. WBC 20-99 x 10 9 /l at diagnosis seemed to be a risk factor as well, though this endpoint only reached statistical significance in the multiple regression analysis (p=0.04). FAB M3 was a risk factor for ED 15-42 or TRD in the multiple Cox regression analysis (11% vs. 5%, p=0.05), but only few patients had this FAB type. Allogeneic HSCT was also associated with a slight excess risk of death from treatment-related complications (HR, 2.0; 95% confidence interval (CI), 0.8-4.9; p=0.1). Sex, platelet

Death in FAB M3

Eight (29%) of 28 patients with FAB type M3 suffered ED or TRD, two (25%) within the first two weeks of diagnosis both from a CNS bleeding before starting chemotherapy or ATRA (Table 4). The other six all died from infections. FAB M3 was not a significant risk factor compared with other FAB types for ED 0-14 (7% vs. 3%, p=0.2), but it was a significant risk factor for ED 15-42 or TRD (11% vs. 5%, p=0.05).

Cause of death

Death from infection:

Infection was the main reason for ED or TRD (n=46; 66%) (Table 5). 7).

Death from cardiac failure:

Only one patient receiving a cumulative dose of doxorubicin of 150 mg/m 2 and mitoxantrone 60 mg/m 2 died from cardiac failure. Six patients dying from infection had cardiac insufficiency a few days preceding their death but in all cases, cardiac failure was considered part of septic shock.

Discussion

The three consecutive NOPHO-AML protocols have shown a successive improvement in outcome from a 5-year overall survival of 38% in the NOPHO-AML-84 to 64% in the NOPHO-AML-93 [START_REF] Lie | Long-term results in children with AML: NOPHO-AML Study Group--report of three consecutive trials[END_REF]. This analysis shows that 13% of children diagnosed with AML in the Nordic countries died before starting treatment or from treatment-related complications. The rates of ED and TRD rose from 11% in NOPHO-AML-84 to 29% in -88, but then fell to 8% in -93. A total of 88% of the patients with ED 0-14 died due to haemorrhage or leukostasis. The risk factors for death before day 15 were hyperleukocytosis, age <2 years or >10 years.

After day 15, infection caused 81% of ED 15-42 and TRDs. Risk factors were age (<2 years or >10 years at diagnosis), WBC 20-99 x 10 9 /l, FAB M3 and treatment according to the NOPHO-AML-88 protocol.

The number of EDs and TRDs has been estimated in different AML trials as shown in 2005). ED occurred in 2-10% and TRD in 2-16%. The percentage was higher with a high rate of HSCT. With a 5-year overall survival of 60% or above, our ED and TRD results resemble those reported by others [START_REF] Riley | Treatment-related deaths during induction and first remission of acute myeloid leukaemia in children treated on the Tenth Medical Research Council acute myeloid leukaemia trial (MRC AML10). The MCR Childhood Leukaemia Working Party[END_REF][START_REF] Creutzig | Early deaths and treatment-related mortality in children undergoing therapy for acute myeloid leukemia: analysis of the multicenter clinical trials AML-BFM 93 and AML-BFM 98[END_REF].

Several groups have reported a decrease in toxic death rates over time without concomitant changes in therapy or even with more intensive therapy. However in the 1982-1997 period, the Dutch childhood oncology group observed that chemotherapyrelated mortality rose from 3% to 8% during consolidation, which was perhaps related to the scheduling of intensification blocks [START_REF] Slats | Causes of death--other than progressive leukemia--in childhood acute lymphoblastic (ALL) and myeloid leukemia (AML): the Dutch Childhood Oncology Group experience[END_REF]. This illustrates the delicate balance between minor changes in treatment and the risk of treatmentrelated mortality. Within the different NOPHO-AML protocols, no significant reduction in the number of EDs and TRDs occurred over time in contrast to the learning curve demonstrated in the CCG-2961 study [START_REF] Lange | Outcomes in CCG-2961, a children's oncology group phase 3 trial for untreated pediatric acute myeloid leukemia: a report from the children's oncology group[END_REF].

The causes of ED and TRD were similar in the different AML studies despite variations in supportive care practices [START_REF] Lehrnbecher | International variations in infection supportive care practices for paediatric patients with acute myeloid leukaemia[END_REF]. In contrast to the excess mortality rate due to cardiac causes (standardized mortality rate=5.0) in the Childhood Cancer Survivor Study (CCSS) [START_REF] Armstrong | Late mortality among 5-year survivors of childhood cancer: a summary from the Childhood Cancer Survivor Study[END_REF], the frequency of cardiac mortality in our population was low.

Three patients died 7-12 years after end of treatment from late effects of treatment or complications in relation to initial AML manifestations, which underlines the importance of continued long-term follow-up. In the CCSS, 4% of 272 5-year AML survivors treated without HSCT died from other causes than relapse 8-26 years after diagnosis [START_REF] Mulrooney | Twenty years of follow-up among survivors of childhood and young adult acute myeloid leukemia: a report from the Childhood Cancer Survivor Study[END_REF].

In the AML-BFM-93 and -98 trials, risk factors for death before day 15 were low performance status at presentation, initial CNS involvement, hyperleukocytosis and FAB M5 [START_REF] Creutzig | Early deaths and treatment-related mortality in children undergoing therapy for acute myeloid leukemia: analysis of the multicenter clinical trials AML-BFM 93 and AML-BFM 98[END_REF]. After day 15, age above 10 years and FAB M0 were risk factors. Further studies are needed to determine whether pharmacokinetic differences account for the increased death rate among children <2 years and >10 years of age [START_REF] Palle | Etoposide pharmacokinetics in children treated for acute myeloid leukemia[END_REF][START_REF] Palle | Thioguanine pharmacokinetics in induction therapy of children with acute myeloid leukemia[END_REF]. We had no data on BMI at diagnosis, but the Children's Cancer Group (CCG)-2961 showed that overweight and underweight children and adolescents with AML were more likely to experience treatment-related mortality [START_REF] Lange | Mortality in overweight and underweight children with acute myeloid leukemia[END_REF]. The high rate of ED and TRD in APL patients in the NOPHO-AML studies may be due to the low incidence of In the AML-BFM-78 and -83 studies, the risk of ED due to leukostasis and/or haemorrhage was significantly increased in children with FAB M5, hyperleukocytosis, and extramedullary organ involvement [START_REF] Creutzig | Early deaths due to hemorrhage and leukostasis in childhood acute myelogenous leukemia. Associations with hyperleukocytosis and acute monocytic leukemia[END_REF]. These findings are in line with the results from our studies. We also found a higher proportion of EDs from leukostasis/haemorrhage in children <2 years than in children between 2 and 9 years of age. We observed a small decline in the incidence of hyperleukocytosis over time from 18% in the NOPHO-AML-84, 19% in the NOPHO-AML-88 to 13% in the NOPHO-AML-93 protocol. The low number of EDs in the NOPHO-AML-84 (3%) and -93 (2%) precludes any conclusion as to whether the lower frequency of hyperleukocytosis reduced ED as shown in the study from St. Jude Children's Research Hospital [START_REF] Inaba | Clinical and biologic features and treatment outcome of children with newly diagnosed acute myeloid leukemia and hyperleukocytosis[END_REF].

Next to relapses, infection remains the most important cause of morbidity and mortality among children with AML. Infections caused 85% of all EDs and TRDs when deaths before starting treatment were excluded. The infection-related mortality of 9% (46/525) is comparable to that reported in other recent AML studies quoting frequencies of 5-11% [START_REF] Riley | Treatment-related deaths during induction and first remission of acute myeloid leukaemia in children treated on the Tenth Medical Research Council acute myeloid leukaemia trial (MRC AML10). The MCR Childhood Leukaemia Working Party[END_REF][START_REF] Lehrnbecher | Infectious complications in pediatric acute myeloid leukemia: analysis of the prospective multi-institutional clinical trial AML-BFM 93[END_REF][START_REF] Brunet | Low incidence of sepsis due to viridans streptococci in a ten-year retrospective study of pediatric acute myeloid leukemia[END_REF][START_REF] Sung | Microbiologically documented infections and infection-related mortality in children with acute myeloid leukemia[END_REF]. However, it is difficult to compare results and draw conclusions about trends in infection-related mortality due to the use of different definitions and underestimation of the true incidence of these infections. We had a large amount of infection-related deaths where no microorganism could be identified. Like in other studies, a significant number of invasive fungal infections were diagnosed only at autopsy [START_REF] Groll | Trends in the postmortem epidemiology of invasive fungal infections at a university hospital[END_REF]. Risk factors for infection-related deaths in the CCG 2961 trial were underweight, non-white race and age above 16 years [START_REF] Sung | Microbiologically documented infections and infection-related mortality in children with acute myeloid leukemia[END_REF]. In our study, age <2 or >10 years at diagnosis were risk factors for death due to infection. The NOPHO-AML-88 used the principle of timed sequential induction which caused an unacceptable increase in ED and TRD. This is similar to the results from the CCG 2891 trial, where fatal infections were more common during intensive than during standard timed induction [START_REF] Sung | Infections and association with different intensity of chemotherapy in children with acute myeloid leukemia[END_REF].

To allow comparison between cooperative AML groups and to improve our understanding of infection outcomes, standard infection definitions should be used [START_REF] Sung | Infections in pediatric acute myeloid leukemia: Lessons learned and unresolved questions[END_REF]. To reduce infection-related mortality, it is important to improve both prevention and treatment through prophylaxis, early diagnosis and aggressive management. The current NOPHO-AML 2004 protocol includes prophylactic anti-fungal therapy with fluconazole but no prophylactic G-CSF or anti-bacterial therapy since there is no solid evidence that these measures reduce mortality in children with AML. However, a recent study from St. Jude Children's Research Hospital reduced the odds of bacterial sepsis by 93% using intravenous vancomycin with oral ciprofloxacin or a cephalosporin [START_REF] Kurt | Prophylactic antibiotics reduce morbidity due to septicemia during intensive treatment for pediatric acute myeloid leukemia[END_REF]. A relatively small number of patients were studied and larger multiinstitutional studies are needed before recommendations on these prophylactic antibiotic regimens can be made. The development of targeted and more leukaemia-specific drugs may reduce the toxic effect on normal cells and thereby reduce infection-related morbidity. Identification of possible immunogenetic risk profiles at the start of therapy could potentially be helpful for risk-adapted and individualized supportive care [START_REF] Hartel | Current approaches for risk stratification of infectious complications in pediatric oncology[END_REF]. Initial data have demonstrated that IL-6 and chitotriosidase polymorphisms may modify infection risk [START_REF] Lehrnbecher | Common genetic variants in the interleukin-6 and chitotriosidase genes are associated with the risk for serious infection in children undergoing therapy for acute myeloid leukemia[END_REF]. In a study from the CCG, polymorphisms in the gene encoding cytidin deaminase were associated with an increased risk of treatment-related mortality with cytarabine-based therapy for AML [START_REF] Bhatla | Cytidine deaminase genotype and toxicity of cytosine arabinoside therapy in children with acute myeloid leukemia[END_REF].

Our study has some limitations. AML is rare in children and the relatively low number of patients therefore hampers statistical analysis of risk factors at subgroup level.

Limited information in the medical records sometimes made it difficult to categorize the cause of death. In some cases, it was difficult to assess whether the patient was in complete remission, and to determine whether the cause of death was treatmentor disease-related.

The number of EDs and TRDs in childhood AML in the Nordic countries remains substantial. Further studies should be conducted to explore the pathogenesis behind leukostasis/haemorrhage and prospective studies of the effect of cytoreduction should be performed. Optimal antifungal prophylaxis is essential, and studies on the benefit of antibacterial prophylaxis and individual risk factors for ED and TRD are needed.

  Since July 1984, all children diagnosed with AML in the Nordic countries (Denmark, Finland, Iceland, Norway and Sweden) have been registered and treated on common protocols conducted by the NOPHO-AML Study Group (Nordic Society of Paediatric Haematology and Oncology). The study is population-based for patients younger than 15 years of age, whereas 15-18-years-old patients are enrolled according to local practice. Patients diagnosed from 1 July 1984 to 31 December 2003 were identified in the database and included in the present historical prospective cohort study. Excluded were patients treated according to other protocols than NOPHO-

  patients registered as non-responders were reviewed to verify the correctness of the data. Leukaemia-related data such as white blood cell count (WBC), platelet count and presence of CNS leukaemia at diagnosis, FAB-type and cytogenetics were collected from the database. Performance status at diagnosis was not available. NOPHO-AML-84/88/93 treatment The treatment regimens of the NOPHO-AML trials are shown in Figure I and the treatment elements including cumulative drug doses are summarised in Tables

  were compared by Chi-square or Fischer's exact tests.

  The p-values reported are two-tailed, and p-values less than 0.05 were considered significant. The statistical analyses were performed using Stata Statistical Software, Release 10 (College Station, TX: StataCorp 2007).

  countries, 13% (70/525) of children diagnosed with AML died before starting treatment or from treatment-related complications (Table

  of treatment. She had received a heart transplant seven years prior to her death due to anthracycline-induced cardiomyopathy. A 17-year-old boy had candidiasis in the liver and spleen during induction. Later, liver cirrhosis with oesophageal varices developed. He died from multiorgan failure seven years after end of treatment, following a month with symptoms of pulmonary infection. A seven months old girl with AML FAB M4, WBC 170 x 10 9 /l and platelets 75 x 10 9 /l presented at diagnosis with cerebral bleeding and died twelve years later as a consequence of the severe brain damage. Risk factors for ED 15-42 or TRD Both univariate and multiple Cox regression analyses of the composite endpoints consisting of ED 15-42 or TRD showed that age at diagnosis was a significant prognostic factor (Table

  of CNS leukaemia at diagnosis, cytogenetics and size of the treating department were not statistically significant risk factors.

  After day 15, infection was considered the cause of death in 44 (81%) of 54 deaths. Infection was the cause of death in eight (80%) of ten patients dying from treatment-related causes during HSCT.In fourteen patients, fatal complications caused by infections occurred as a result of bacterial pathogens, in six cases a gram-positive isolate and in eight a gramnegative isolate. Pseudomonas aeruginosa (n=7) were predominant among gramnegative pathogens, Klebsiella was isolated in a single case. The gram-positive pathogens were Streptococci (n=2), Staphylococci (n=2), Bacillus cereus (n=1) and Gemella (n=1). Eleven patients died of fungal infection. This was caused by Aspergillus in six patients, by Candida in four patients and in one case no specific microorganism could be identified. Nine (82%) of 11 cases of fungal infection were only diagnosed by autopsy. Viruses causing TRD were CMV (n=1) n=1). In four cases, the infection was polybacterial or -microbial. Fifteen patients died of clinically/radiologically documented infections without having a pathogen identified.Age <2 (p=0.02) or >10 years (p=0.01) at diagnosis were the only statistically significant risk factors for infection-related ED 15-42 or TRD (Table

  Northern Europe and therefore limited experience and late introduction of ATRA.

  Figure legend

  Figure 1.

  Abbreviations: ED, early death; TRD, treatment-related death; CR, complete remission; NR, non-responders

Table 8 (

 8 [START_REF] Tsukimoto | Risk-stratified therapy and the intensive use of cytarabine improves the outcome in childhood acute myeloid leukemia: the AML99 trial from the Japanese Childhood AML Cooperative Study Group[END_REF][START_REF] Riley | Treatment-related deaths during induction and first remission of acute myeloid leukaemia in children treated on the Tenth Medical Research Council acute myeloid leukaemia trial (MRC AML10). The MCR Childhood Leukaemia Working Party[END_REF][START_REF] Creutzig | Early deaths and treatment-related mortality in children undergoing therapy for acute myeloid leukemia: analysis of the multicenter clinical trials AML-BFM 93 and AML-BFM 98[END_REF][START_REF] Rubnitz | Death during induction therapy and first remission of acute leukemia in childhood: the St. Jude experience[END_REF][START_REF] Slats | Causes of death--other than progressive leukemia--in childhood acute lymphoblastic (ALL) and myeloid leukemia (AML): the Dutch Childhood Oncology Group experience[END_REF] Ribeiro et al, 2005; Gibson et al, 2005; Creutzig et al, 2006; Kardos et al, 2005; Ravindranath et al, 2005; Kaspers & Creutzig, 2005; Smith et al, 2005; Entz-Werle et al, 2005; Perel et al, 2005; Dluzniewska et al, 2005; Pession et al,

Table 1 .

 1 NOPHO-AML-84/88/93 treatment elements

	ATDox	Cytarabine 100 mg/m	2 /day continuous infusion day 1-4
		6-Thioguanine 100 mg/m	2 orally every 12 h day 1-4
		Doxorubicin 75 mg/m 2 8-hour infusion day 5
	HA2	Cytarabine 2 g/m	2 2-hour infusion every 12 h day 1-3
	ATEDox	Cytarabine 200 mg/m	2 /day continuous infusion day 1-4
		6-Thioguanine 100 mg/m	2 orally every 12 h day 1-4
		Etoposide 100 mg/m	2 /day continuous infusion day 1-4
		Doxorubicin 75 mg/m 2 8-hour infusion day 5
	AM	Cytarabine 100 mg/m	2 /day continuous infusion day 1-5
		Mitoxantrone 10 mg/m	2 30-minute infusion day 1-3
	HA1M	Cytarabine 1 g/m	2 2-hour infusion every 12 h day 1-3
		Mitoxantrone 10 mg/m	2 30-minute infusion day 3-5
	HA2E	Cytarabine 2 g/m	2 2-hour infusion every 12 h day 1-3
		Etoposide 100 mg/m	2 /day 1-hour infusion day 2-5
	HA3	Cytarabine 3 g/m	2 2-hour infusion every 12 h day 1-3
	CNS therapy	Methotrexate age-adjusted 6-12 mg intrathecal on the first day of all courses
	For children less than 2 years of age, drug doses were calculated according to body weight with 1 m	2 equalling 30
	kg

Table 2 .

 2 NOPHO-AML-84/88/93 cumulative doses of cytarabine, etoposide and anthracyclines

		Cytarabine	Etoposide	Doxorubicin / Mitoxantrone
						Cumulative dose of
						anthracyclines*
		(g/m	2 )	(mg/m	2 )	(mg/m	2 )
	NOPHO-AML-84	50.4	0		225 / 0
						Cum. dose = 225
	NOPHO-AML-88	50.1	1600	150 / 60
						Cum. dose = 450
	NOPHO-AML-93	GR: 49.6	GR: 1600	GR: 150 / 30
		PR: 61.3	PR: 1600	PR: 75 / 60
						Cum. dose GR = 300, PR = 375
	Abbreviations: GR, good responders; PR, poor responders
	* Calculated applying the following conversion factors to daunorubicin-equivalents: doxorubicin 1 x, mitoxantrone 5
	x

Table 3 .

 3 Number of early deaths, treatment-related deaths, non-responders and relapses in NOPHO-AML-84/88/93 protocols Status

		%
	Total	No. of patients
	NOPHO AML-93	No. of patients %
	NOPHO AML-88	No. of patients %
	NOPHO AML-84	No. of patients %
	Period
		No. of patients

Table 7 .

 7 Risk factors for early death day 15 -42 or treatment-related death caused by infection in NOPHO-AML-84/88/93 protocols Abbreviations: ED15-42, early death day 15-42; TRD, treatment-related death; HR, hazard ratio; 95% CI, 95% confidence interval * Adjusted for age (2-9 vs. <2 or >10 years) and protocol (NOPHO-AML-84 vs. NOPHO-AML-88 or NOPHO-AML-93)

	Risk factors	All patients	ED15-42 or TRD	HR	Adjusted HR*
				caused by
				infection	(95% CI)	(95% CI)
		n=525	%	n=46	%	Simple	Multiple
	Sex
	Female	272	52	26	57	1.2 (0.7-2.2)	1.2 (0.7-2.2)
	Male	253	48	20	43	1.0	1.0
	Age (years)
	<2	141	27	16	35	2.9 (1.3-6.6)	2.8 (1.2-6.3)
	2-9	215	41	9	19	1.0	1.0
	>10 (10-14)	169	32	21 (21)	46	3.1 (1.4-6.9)	3.1 (1.4-6.7)
	Leukocytes at diagnosis (x10	9 /l)
	<20	285	54	23	50	1.0	1.0
	20-99	159	30	18	39	1.5 (0.8-2.7)	1.6 (0.9-2.9)
	>100	81	16	5	11	0.9 (0.4-2.4)	0.7 (0.3-2.0)
	Protocol
	NOPHO-AML-84	96	18	8	17	1.0	1.0
	NOPHO-AML-88	114	22	20	44	2.2 (1.0-5.1)	2.1 (0.9-4.7)
	NOPHO-AML-93	315	60	18	39	0.6 (0.3-1.5)	0.6 (0.3-1.4)

British Journal of Haematology

Acknowledgements

We are grateful to professor M.Vaeth, Department of Biostatistics, University of Aarhus for excellent statistical assistance.

We thank all principal investigators of the NOPHO-AML-84/88/93 studies and the Nordic paediatric oncology centres for providing patient files for this study.

Investigators in Denmark: S.Rosthøj/E.Oestergaard, Aalborg; N.Clausen/

The A. P. Møller Foundation for the Advancement of Medical Science. The Aarhus University Hospital Research Initiative Foundation. The Aase and Ejnar Danielsen Foundation. The Anders Hasselbalch Foundation. The Bent Bøgh and wife Inge Bøgh Foundation. The Carl J. Beckers Foundation. The Dagmar Marshall Foundation. The Danish Cancer Society. The Danish Childhood Cancer Foundation.

The Danish Graduate School in Clinical Oncology. The Eva and Henry Fraenkel Foundation. The Frode V. Nyegaard and Wife Foundation. The Johannes Fogh-Nielsen and wife Ella Fogh-Nielsen Grant. The Karen Elise Jensen Foundation.

Authorship

Lene Molgaard-Hansen participated in the design of the study, provided clinical data, handled the data, performed the statistical analysis and drafted the manuscript. Henrik Hasle participated in the design of the study, participated in the interpretation of the data and the drafting of the manuscript.

Merja Möttönen, Heidi Glosli, Guðmundur K Jónmundsson and Jonas Abrahamsson provided clinical data in their capacity as national coordinators of this study and participated in the interpretation of the data.

All authors read and approved the final manuscript. 3) Abbreviations: ED15-42, early death day 15-42; TRD, treatment-related death; HR, hazard ratio; 95% CI, 95% confidence interval; HSCT, haematopoietic stem cell transplantation * Adjusted for age (2-9 vs. <2 or >10 years), leukocytes at diagnosis (<20 vs. 20-99 or >100 x 10 9 /l) and protocol (NOPHO-AML-84 vs. NOPHO-AML-88 or NOPHO-AML-93) † Favourable cytogenetics: t(8;21)(q22;q22), t(9;11)(p22;q23), t(15;17)(q22;q12-21), inv16(p13q22) and t(16;16)(p13;q22)

Survival after diagnosis

Age at diagnosis

Sex

WBC at diagnosis

Platelets at diagnosis

Cytogenetics