A Semi-Mechanistic Population Pharmacokinetic/Pharmacodynamic Model of Bortezomib in Pediatric Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia.

INTRODUCTION: The pharmacokinetics (PK) of the 20S proteasome inhibitor bortezomib are characterized by a large volume of distribution and a rapid decline in plasma concentrations within the first hour after administration. An increase in exposure was observed in the second week of treatment, which has previously been explained by extensive binding of bortezomib to proteasome in erythrocytes and peripheral tissues. We characterized the nonlinear population PK and pharmacodynamics (PD) of bortezomib in children with acute lymphoblastic leukemia. METHODS: Overall, 323 samples from 28 patients were available from a pediatric clinical study investigating bortezomib at an intravenous dose of 1.3 mg/m2 twice weekly (Dutch Trial Registry number 1881/ITCC021). A semi-physiological PK model for bortezomib was first developed; the PK were linked to the decrease in 20S proteasome activity in the final PK/PD model. RESULTS: The plasma PK data were adequately described using a two-compartment model with linear elimination. Increased concentrations were observed in week 2 compared with week 1, which was described using a Langmuir binding model. The decrease in 20S proteasome activity was best described by a direct effect model with a sigmoidal maximal inhibitory effect, representing the relationship between plasma concentrations and effect. The maximal inhibitory effect was 0.696 pmol AMC/s/mg protein (95% confidence interval 0.664-0.728) after administration. CONCLUSION: The semi-physiological model adequately described the nonlinear PK and PD of bortezomib in plasma. This model can be used to further optimize dosing of bortezomib.

Genotype-outcome correlations in pediatric AML: the impact of a monosomal karyotype in trial AML-BFM 2004.

We conducted a cytogenetic analysis of 642 children with de novo acute myeloid leukemia (AML) treated on the AML-Berlin-Frankfurt-Münster (BFM) 04 protocol to determine the prognostic value of specific chromosomal aberrations including monosomal (MK+), complex (CK+) and hypodiploid (HK+) karyotypes, individually and in combination. Multivariate regression analysis identified in particular MK+ (n=22) as a new independent risk factor for poor event-free survival (EFS 23±9% vs 53±2% for all other patients, P=0.0003), even after exclusion of four patients with monosomy 7 (EFS 28±11%, P=0.0081). CK+ patients without MK had a better prognosis (n=47, EFS 47±8%, P=0.46) than those with MK+ (n=12, EFS 25±13%, P=0.024). HK+ (n=37, EFS 44±8% for total cohort, P=0.3) influenced outcome only when t(8;21) patients were excluded (remaining n=16, EFS 9±8%, P<0.0001). An extremely poor outcome was observed for MK+/HK+ patients (n=10, EFS 10±10%, P<0.0001). Finally, isolated trisomy 8 was also associated with low EFS (n=16, EFS 25±11%, P=0.0091). In conclusion, monosomal karyotype is a strong and independent predictor for high-risk pediatric AML. In addition, isolated trisomy 8 and hypodiploidy without t(8;21) coincide with dismal outcome. These results have important implications for risk stratification and should be further validated in independent pediatric cohorts.

Genomic and transcriptional landscape of P2RY8-CRLF2-positive childhood acute lymphoblastic leukemia.

Children with P2RY8-CRLF2-positive acute lymphoblastic leukemia have an increased relapse risk. Their mutational and transcriptional landscape, as well as the respective patterns at relapse remain largely elusive. We, therefore, performed an integrated analysis of whole-exome and RNA sequencing in 41 major clone fusion-positive cases including 19 matched diagnosis/relapse pairs. We detected a variety of frequently subclonal and highly instable JAK/STAT but also RTK/Ras pathway-activating mutations in 76% of cases at diagnosis and virtually all relapses. Unlike P2RY8-CRLF2 that was lost in 32% of relapses, all other genomic alterations affecting lymphoid development (58%) and cell cycle (39%) remained stable. Only IKZF1 alterations predominated in relapsing cases (P=0.001) and increased from initially 36 to 58% in matched cases. IKZF1's critical role is further corroborated by its specific transcriptional signature comprising stem cell features with signs of impaired lymphoid differentiation, enhanced focal adhesion, activated hypoxia pathway, deregulated cell cycle and increased drug resistance. Our findings support the notion that P2RY8-CRLF2 is dispensable for relapse development and instead highlight the prominent rank of IKZF1 for relapse development by mediating self-renewal and homing to the bone marrow niche. Consequently, reverting aberrant IKAROS signaling or its disparate programs emerges as an attractive potential treatment option in these leukemias.

Randomised Introduction of 2-CDA as Intensification during Consolidation for Children with High-risk AML--results from Study AML-BFM 2004.

BACKGROUND: The outcome in children and adolescents with high-risk (HR) acute myeloid leukemia (AML) is still unsatisfactory. Therefore, in study AML-BFM 2004 we aimed to improve outcome of HR-patients by adding moderately dosed 2-Chloro-2-Deoxyadenosine (2-CDA) to the respective consolidation treatment backbone without increasing toxicity. The aim was to improve prognosis especially in FAB M4/M5/MLL patients, who represent the largest subgroup of HR patients. PATIENTS AND METHODS: In total, 343 children and adolescents with HR-AML were randomized to receive or not 2-CDA (6 mg/m²/d, days 1, 3) in combination with cytarabine/idarubicine (AI=500 mg/m² cytarabine 5 days continuous infusion plus 7 mg/m²/d idarubicin, days 3 and 5). RESULTS: RESULTS for patients of the AI/2-CDA arm (n=168) vs. the AI-arm (n=175) were similar: 5-year overall survival 68±4 vs. 72±4%, plogrank=0.38, event-free survival 53±4 vs. 49±4%, plogrank=0.77; cumulative incidence of relapse at 5 years: 35±4 vs. 37±4%, p(Gray)=0.89. RESULTS in patients with MLL rearrangement or FAB M4/M5 were also similar in the treatment groups. In addition, toxicities did not differ between the two arms. CONCLUSION: We conclude that additional, moderate dose 2-CDA does not improve prognosis in HR-patients when given during consolidation treatment. Its effect might be too low in this multidrug regimen, where the strongest effects are achieved during induction, or the chosen dose of 2-CDA might have been too low.

KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia.

High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18-30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.

Monitoring minimal residual disease in children with high-risk relapses of acute lymphoblastic leukemia: prognostic relevance of early and late assessment.

The prognosis for children with high-risk relapsed acute lymphoblastic leukemia (ALL) is poor. Here, we assessed the prognostic importance of response during induction and consolidation treatment prior to hematopoietic stem cell transplantation (HSCT) aiming to evaluate the best time to assess minimal residual disease (MRD) for intervention strategies and in future trials in high-risk ALL relapse patients. Included patients (n=125) were treated uniformly according to the ALL-REZ BFM (Berlin-Frankfurt-Münster) 2002 relapse trial (median follow-up time=4.8 years). Patients with MRD ⩾10(-3) after induction treatment (76/119, 64%) or immediately preceding HSCT (19/71, 27%) had a significantly worse probability of disease-free survival 10 years after relapse treatment begin, with 26% (±6%) or 23% (±7%), respectively, compared with 58% (±8%) or 48% (±7%) for patients with MRD <10(-3). Conventional intensive consolidation treatment reduced MRD to <10(-3) before HSCT in 63% of patients, whereas MRD remained high or increased in the rest of this patient group. Our data support that MRD after induction treatment can be used to quantify the activity of different induction treatment strategies in phase II trials. MRD persistence at ⩾10(-3) before HSCT reflects a disease highly resistant to conventional intensive chemotherapy and requiring prospective controlled investigation of new treatment strategies and drugs.

Effective childhood cancer treatment: the impact of large scale clinical trials in Germany and Austria.

In Germany and Austria, more than 90% of pediatric cancer patients are enrolled into nationwide disease-specific first-line clinical trials or interim registries. Essential components are a pediatric cancer registry and centralized reference laboratories, imaging review, and tumor board assistance. The five-year overall survival rate in countries where such infrastructures are established has improved from <20% before 1950 to >80% since 1995. Today, treatment intensity is tailored to the individual patient's risk to provide the highest chances of survival while minimizing deleterious late effects. Multicenter clinical trials are internationalized and serve as platforms for further improvements by novel drugs and biologicals.

Prognostic value of genetic alterations in children with first bone marrow relapse of childhood B-cell precursor acute lymphoblastic leukemia.

Despite risk-adapted treatment, survival of children with relapse of acute lymphoblastic leukemia (ALL) remains poor compared with that of patients with initial diagnosis of ALL. Leukemia-associated genetic alterations may provide novel prognostic factors to refine present relapse treatment strategies. Therefore, we investigated the clinical relevance of 13 recurrent genetic alterations in 204 children treated uniformly for relapsed B-cell precursor ALL according to the ALL-REZ BFM 2002 protocol. The most common alterations were deletions of CDKN2A/2B, IKZF1, PAX5, ETV6, fusion of ETV6-RUNX1 and deletions and/or mutations of TP53. Multivariate analysis identified IKZF1 deletion and TP53 alteration as independent predictors of inferior outcome (P=0.002 and P=0.001). Next, we investigated how both alterations can improve the established risk stratification in relapsed ALL. Intermediate-risk relapse patients with low minimal residual disease are currently considered to have a good prognosis. In this group, deletion of IKZF1 and alteration of TP53 identify patients with significantly inferior outcome (P<0.001). In high-risk relapse patients, deletion of IKZF1 is strongly predictive of a second relapse after stem cell transplantation (P<0.001). We conclude that IKZF1 and TP53 represent relevant prognostic factors that should be considered in future risk assessment of children with relapsed ALL to indicate treatment intensification or intervention.

[GATA1-mutation associated leukemia in children with trisomy 21 mosaic]. / GATA1-Mutations-assoziierte Leukämien bei Kindern mit Trisomie 21-Mosaik.

Mutations of the hematopoietic transcription factor GATA1 (GATA1s) are pathognomonic in newborn with transient leukemia and children with Down syndrome and myeloid leukemia (ML-DS). Both TL and ML-DS can also occur in children with trisomy 21 mosaic.Between 2002 and 2011, 15 newborns and infants were diagnosed with DS mosaic. 9 of them presented with TL and 8 children suffered from ML-DS; 2 of them with a history of TL. In children without stigmata the special morphology and immunophenotype of blasts triggered the screening for GATA1 mutation and trisomy 21 mosaic.All newborns with TL achieved complete remission (CR). Due to clinical symptoms caused by the leukemic blasts, in 3 children low-dose cytarabine was applied. 1 patient died due to cardiac defect. In all patients GATA 1 s was confirmed. 6 children with ML-DS were initially treated according the AML-BFM protocol. After ML-DS was confirmed, therapy was continued with the intensity reduced schedule according to the ML-DS 2006 protocol. All children are still in CR (follow-up 1.8-7 years, median 2.7 yrs). 2 children with unknown trisomy 21 mosaic were diagnosed as acute megakaryoblastic leukemia (AMKL) and treated according the high risk arm of the AML-BFM 2004 including allogeneic stem cell transplantation in one child). GATA1 mutation was identified retrospectively. Both children are alive in CR.GATA1s associated leukemia has to be excluded in all young children with AMKL (<5 years old) to prevent overtreatment. Treatment with reduced intensity seems sufficient in children trisomy 21 mosaic and ML-DS.

Favorable outcome in infants with AML after intensive first- and second-line treatment: an AML-BFM study group report.

Infants <1 year of age have a high prevalence of prognostically unfavorable leukemias and a presumed susceptibility to treatment-related toxicities. A total of 125 infants with acute myeloid leukemia (AML) were treated in studies AML-BFM-98 (n = 59) and -2004 (n = 66). Treatment regimens of both studies were comparable, consisting of intensive induction followed by four courses (mainly high-dose cytarabine and anthracyclines). Allogeneic-hematopoietic stem-cell-transplantation (allo-HSCT) in 1st remission was optional for high-risk (HR) patients. Most infants (120/125=96%) were HR patients according to morphological, cytogenetic/molecular genetic and response criteria. Five-year overall survival was 66 ± 4%, and improved from 61 ± 6% in study-98 to 75 ± 6% in study-2004 (P(logrank) 0.14) and event-free survival rates were 44 ± 6% and 51 ± 6% (P(logrank) 0.66), respectively. Results in HR infants were similar to those of older HR children (1-<2- or 2-<10-year olds, P(logrank) 0.90 for survival). Survival rates of HSCT in 1st remission, initial partial response and after relapse were high (13/14, 2/8 and 20/30 patients, respectively). The latter contributes to excellent 5-year survival after relapse (50±8%). Despite more severe infections and pulmonary toxicities in infants, treatment-related death rate was identical to that of older children (3%). Our data indicate that intensive frontline and relapse AML treatment is feasible in infants, toxicities are manageable, and outcome is favorable.

Autologous purified peripheral blood SCT in childhood low-risk relapsed ALL.

The treatment of childhood B-cell-precursor ALL after isolated-extramedullary or late relapse is controversial. Most approaches are based on chemotherapy or allogeneic transplantation. The aim of this report is to assess the long-term outcome of children with 'low-risk' relapsed ALL treated according to a prospective purified auto-transplantation protocol. From January 1997 to March 2004, at a single pediatric Center, 30 ALL consecutive children, lacking an HLA-identical sibling, were treated according to the autologous purified peripheral blood stem cell protocol after isolated-extramedullary (7) or late medullary (24) relapse. After the 'DIAVE' mobilizing regimen a median of 11.6 × 10(6)CD34+/Kg (range 3.9-27.4) were collected. Leukaphereses were depleted by 99% of CD19+cells (range 98-100) by means of a double step immunological purification. The conditioning regimen included TBI. No early severe complications nor transplant-related deaths occurred; late effects, as expected, mostly consisted in endocrinological issues and were assessed at a median follow-up of 8.5 years. Five-year-EFS and survival were 68.5% (s.e. 7.9) and 85.7% (s.e. 5.9), respectively, for the 35 eligible patients and 70.0% (s.e. 8.4) and 86.7% (s.e. 6.2) for the 30 patients actually transplanted as per protocol. The outcome of this series favorably compares with historical data regarding both autologous transplantation and standard salvage chemotherapy.

Standardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008.

Assessment of minimal residual disease (MRD) has acquired a prominent position in European treatment protocols for patients with acute lymphoblastic leukemia (ALL), on the basis of its high prognostic value for predicting outcome and the possibilities for implementation of MRD diagnostics in treatment stratification. Therefore, there is an increasing need for standardization of methodologies and harmonization of terminology. For this purpose, a panel of representatives of all major European study groups on childhood and adult ALL and of international experts on PCR- and flow cytometry-based MRD assessment was built in the context of the Second International Symposium on MRD assessment in Kiel, Germany, 18-20 September 2008. The panel summarized the current state of MRD diagnostics in ALL and developed recommendations on the minimal technical requirements that should be fulfilled before implementation of MRD diagnostics into clinical trials. Finally, a common terminology for a standard description of MRD response and monitoring was established defining the terms 'complete MRD response', 'MRD persistence' and 'MRD reappearance'. The proposed MRD terminology may allow a refined and standardized assessment of response to treatment in adult and childhood ALL, and provides a sound basis for the comparison of MRD results between different treatment protocols.

Allogeneic hematopoietic SCT in children with ALL: current concepts of ongoing prospective SCT trials.

The definition of indications for allogeneic SCT in children with high-risk (HR) ALL in the first remission or after the first or subsequent relapse depends on biological features, response to treatment and survival after chemotherapy alone. As the results of frontline and relapse protocols are improving over time, there is a strong need for prospective SCT trials, ensuring a well-standardized procedure regarding all relevant components that are potentially responsible for heterogeneity in post-SCT outcome. Therefore, in 2003, the ALL-BFM and the ALL-REZ BFM Study Group initiated a prospective, international, multicenter trial (ALL-SCT-BFM 2003). This trial will now be extended to a larger consortium, trial ALL-SCT-BFM-international (ALL-SCT-BFMi). Strict rules define HLA-typing, donor selection, conditioning regimen, GvHD prophylaxis and therapy as well as standards of supportive care to reduce treatment-related mortality and establish an early GVL effect. Moreover, comprehensive and closely reviewed documentation and serious adverse event reporting shall ensure high study quality. Case-by-case discussions of any fatal or critical course during annual meetings will improve the culture of failure management and lead to modifications of guidelines of supportive care. Finally, the results of these prospective trials will determine the current potential of the different SCT procedures in HR or relapsed childhood ALL.

Results of the first randomized multicentre trial on childhood acute lymphoblastic leukaemia in Russia.

Until 1990, the survival of children with acute lymphoblastic leukaemia (ALL) in Russia was below 10%. To establish a protocol feasible under conditions there, ALL-MB 91 was designed to avoid prolonged bone marrow aplasia, thereby reducing needs for extensive supportive care, blood transfusions, long-lasting hospitalization and costs. High-dose therapies were avoided, anthracycline use was limited and CNS radiation therapy only foreseen in high-risk patients (about 30%). This was randomized against a modified BFM protocol. From 1995 to 2002, 834 patients of age up to 18 years were registered in 10 centres and 713 received after central randomization the allocated risk-stratified treatment. After a median follow-up of 7 years, the event-free survival (EFS) was 67+/-3% on ALL-MB 91 (N=358) vs 68+/-3% on ALL-BFM 90m (N=355). The overall survival (OS) was 71+/-3% vs 74+/-2%, respectively. Anaemia, thrombocytopenia, agranulocytosis >10 days and hospitalization (median 35 vs 68 days) were lower on ALL-MB 91 (P<0.01, N=197). While EFS and OS were similar with both protocols, ALL-MB 91 significantly incurred fewer toxicity and resource requirements and, therefore, has been increasingly used across Russia.