Micafungin twice-a-week for prophylaxis of invasive Aspergillus infections in children with acute lymphoblastic leukaemia: A controlled cohort study.

OBJECTIVES: Invasive Aspergillus infections during the early phase of childhood acute lymphoblastic leukemia (ALL) treatment come with morbidity and mortality. The interaction with vincristine hampers first-line azole prophylaxis. We describe the efficacy of an alternative twice-a-week micafungin regimen for Aspergillus prophylaxis. METHODS: Newly diagnosed paediatric patients with ALL treated according to the ALL-11 protocol received micafungin twice-a-week (9 mg/kg/dose [max. 300 mg]) during the induction course (first 35 days of treatment) as part of routine care. A historical control cohort without Aspergillus prophylaxis was used. During the first consolidation course (day 36-79), standard itraconazole prophylaxis was used in both groups. The percentage of proven/probable Aspergillus infections during the induction/first consolidation course was compared between the cohorts. The cumulative incidence of proven/probable Aspergillus infections was estimated using a competing risk model. For safety evaluation, liver laboratory chemistry values were analysed. RESULTS: A total of 169 and 643 paediatric patients with ALL were treated in the micafungin cohort (median age: 4 years [range 1-17]) and historical cohort (median age: 5 years [range 1-17]). The percentage of proven/probable Aspergillus infections was 1·2% (2/169) in the micafungin cohort versus 5·8% (37/643) in the historical cohort (p=0.013; Fisher's exact test). The differences in estimated cumulative incidence were assessed (p=0·014; Gray's test). Although significantly higher ALT/AST values were reported in the micafungin cohort, no clinically relevant side effects were observed. CONCLUSIONS: Twice-a-week micafungin prophylaxis during the induction course significantly reduced the occurrence of proven/probable Aspergillus infections in the early phase of childhood ALL treatment.

The impact of saliva collection methods on measured salivary biomarker levels.

Saliva diagnostics have become increasingly popular due to their non-invasive nature and patient-friendly collection process. Various collection methods are available, yet these are not always well standardized for either quantitative or qualitative analysis. In line, the objective of this study was to evaluate if measured levels of various biomarkers in the saliva of healthy individuals were affected by three distinct saliva collection methods: 1) unstimulated saliva, 2) chew stimulated saliva, and 3) oral rinse. Saliva samples from 30 healthy individuals were obtained by the three collection methods. Then, the levels of various salivary biomarkers such as proteins and ions were determined. It was found that levels of various biomarkers obtained from unstimulated saliva were comparable to those in chew stimulated saliva. The levels of potassium, sodium, and amylase activity differed significantly among the three collection methods. Levels of all biomarkers measured using the oral rinse method significantly differed from those obtained from unstimulated and chew-stimulated saliva. In conclusion, both unstimulated and chew-stimulated saliva provided comparable levels for a diverse group of biomarkers. However, the results obtained from the oral rinse method significantly differed from those of unstimulated and chew-stimulated saliva, due to the diluted nature of the saliva extract.

Minimal residual disease and outcome characteristics in infant KMT2A-germline acute lymphoblastic leukaemia treated on the Interfant-06 protocol.

BACKGROUND: The outcome of infants with KMT2A-germline acute lymphoblastic leukaemia (ALL) is superior to that of infants with KMT2A-rearranged ALL but has been inferior to non-infant ALL patients. Here, we describe the outcome and prognostic factors for 167 infants with KMT2A-germline ALL enrolled in the Interfant-06 study. METHODS: Univariate analysis on prognostic factors (age, white blood cell count at diagnosis, prednisolone response and CD10 expression) was performed on KMT2A-germline infants in complete remission at the end of induction (EOI; n = 163). Bone marrow minimal residual disease (MRD) was measured in 73 patients by real-time quantitative polymerase chain reaction at various time points (EOI, n = 68; end of consolidation, n = 56; and before OCTADAD, n = 57). MRD results were classified as negative, intermediate (<5∗10-4), and high (≥5∗10-4). RESULTS: The 6-year event-free and overall survival was 73.9% (standard error [SE] = 3.6) and 87.2% (SE = 2.7). Relapses occurred early, within 36 months from diagnosis in 28 of 31 (90%) infants. Treatment-related mortality was 3.6%. Age <6 months was a favourable prognostic factor with a 6-year disease-free survival (DFS) of 91% (SE = 9.0) compared with 71.7% (SE = 4.2) in infants >6 months of age (P = 0.04). Patients with high EOI MRD ≥5 × 10-4 had a worse outcome (6-year DFS 61.4% [SE = 12.4], n = 16), compared with patients with undetectable EOI MRD (6-year DFS 87.9% [SE = 6.6], n = 28) or intermediate EOI MRD <5 × 10-4 (6-year DFS 76.4% [SE = 11.3], n = 24; P = 0.02). CONCLUSION: We conclude that young age at diagnosis and low EOI MRD seem favourable prognostic factors in infants with KMT2A-germline ALL and should be considered for risk stratification in future clinical trials.

Study protocol: DexaDays-2, hydrocortisone for treatment of dexamethasone-induced neurobehavioral side effects in pediatric leukemia patients: a double-blind placebo controlled randomized intervention study with cross-over design.

BACKGROUND: Dexamethasone, a highly effective drug in treating pediatric acute lymphoblastic leukemia (ALL), can induce serious neurobehavioral side effects. These side effects are experienced by patients and parents as detrimental with respect to health related quality of life (HRQoL). Based on previous studies, it has been suggested that neurobehavioral side effects are associated to cortisol depletion of the mineralocorticoid receptor in the brain. Our previously reported randomized controlled trial, the Dexadagen study (NTR3280), suggests that physiological hydrocortisone addition during dexamethasone treatment may overcome clinically relevant neurobehavioral problems in patients who experience these problems during dexamethasone treatment. With our current study, we aim to replicate these results in a targeted larger sample before further implementing this intervention into standard of care. METHODS: In a national center setting, pediatric ALL patients between 3 and 18 years are enrolled in an Identification study, which identifies patients with clinically relevant dexamethasone-induced neurobehavioral side effects using the Strengths and Difficulties Questionnaire (SDQ). Contributing factors, such as genetic susceptibility, dexamethasone pharmacokinetics as well as psychosocial and family factors are studied to determine their influence in the inter-patient variability for developing dexamethasone-induced neurobehavioral side effects. Patients with clinically relevant problems (i.e. a rise of ≥ 5 points on the SDQ Total Difficulties Score after 5 days of dexamethasone) are subsequently included in a randomized double-blind placebo-controlled trial with a cross-over design. They receive two courses placebo followed by two courses hydrocortisone during dexamethasone treatment, or vice versa, each time at least 16 days without study medication in between. The primary endpoint is change in SDQ score. The secondary endpoints are sleep (measured with actigraphy and the Sleep Disturbance Scale for Children) and HRQoL (Pediatric Quality of Life Questionnaire). DISCUSSION: The results of our current study may contribute to the management of future ALL patients who experience dexamethasone-induced neuropsychological problems as it may improve HRQoL for patients who suffer most from dexamethasone-induced neurobehavioral side effects. Furthermore, by investigating multiple risk factors that could be related to inter-patient variability in developing these side effects, we might be able to identify and treat patients who are at risk earlier during treatment. TRIAL REGISTRATION: Medical Ethical Committee approval number: NL62388.078.17. Affiliation: Erasmus Medical Centre. Netherlands Trial Register: NL6507 ( NTR6695 ). Registered 5 September 2017.

Ten Bird Species, Six Guilds, Three Habitats, and 59 Chlorinated and Brominated POPs: What do 64 Eggs from the Largest Economic Hub of Southern Africa tell us?

There is little information on how POPs in eggs of different terrestrial, wetland, and aquatic birds share a large urban and rural landscape relate. We collected and analysed 64 eggs belonging to ten species of six feeding guilds, and compared organic chlorinated pesticide (OCP), polychlorinated biphenyl (PCB), and brominated flame retardants (BFR) residue concentrations and compositions. The eggs were collected in the Gauteng and the northern part of the Free Sate provinces of South Africa, one of the largest economic hubs in Africa. White-breasted Cormorant and African Darter eggs (at the highest trophic level as large aquatic predators) had the highest ΣOCP and ΣPCB concentrations, and Cape Sparrow and Southern Masked Weaver (granivores) eggs had the lowest concentrations, corresponding to the lowest trophic level in our collection. The highest percentage p,p'-DDT were in eggs of the terrestrial insectivore Crowned Lapwing (24%) and the scavenging African Sacred Ibis (17%), and the lowest in African Darter (1.0%) and White-breasted Cormorant (0.9%) eggs, suggesting that recency of DDT releases in a region cannot be gauged by this metric. African Sacred Ibis and Southern Masked Weaver eggs had the highest ΣBFR concentrations, with Crowned Lapwing, Cattle Egret, and White-breasted Cormorant eggs the least. Based on feeding guilds, the mean ΣPOP concentrations increased from granivore, aquatic omnivore, scavenger, terrestrial insectivore, small aquatic predator, to large aquatic predator. Mean ΣPOP concentrations in eggs increased from terrestrial, to wetland, to aquatic habitat birds. Interesting patterns were observed with multivariate analyses. There were no significant regressions between egg size and any summed POP classes. ΣBFR concentrations were not correlated with ΣOCPs or ΣPCBs. Eggshell thinning of African Darter eggs was associated with p,p'-DDE and ΣPCB suggesting risk. Other metrics also suggest risk. Therefore, different species of terrestrial and aquatic birds from the same area acquire and deposit POPs in different proportions and quantities in their eggs. Trophic levels and habitat explain the overall patterns, but detailed differences were found, some of which we are unable to explain. Based on POPs residues in terrestrial, wetland, and aquatic bird eggs, different POPs classes behave differently in a shared large inland industrial area, complicating deductions about POPs and associated risks based on one or few species.

The evaluation of red blood cell folate and methotrexate levels during protocol M in childhood acute lymphoblastic leukemia.

BACKGROUND: After High-Dose Methotrexate (HD-MTX), folinic acid rescue therapy (Leucovorin) is administered to reduce side effects in pediatric acute lymphoblastic leukemia (ALL) patients. Leucovorin and MTX are structural analogues, possibly competing for cellular transport and intracellular metabolism. We hypothesize that Leucovorin accumulates during consecutive courses, which might result in a lower MTX uptake. METHODS: We prospectively measured red blood cell (RBC) folate and MTX levels during four HD-MTX and Leucovorin courses in 43 patients treated according the DCOG ALL-11 protocol with 2-weekly HD-MTX (5 g/m2/dose) and Leucovorin (15 mg/m2/dose) using LC-MS/MS. We estimated a linear mixed model to assess the relationship between these variables over time. RESULTS: Both RBC MTX-PG and folate levels increased significantly during protocol M. MTX-PG2-5 levels increased most substantially after the first two HD-MTX courses (until median 113.0 nmol/L, IQR 76.8-165.2) after which levels plateaued during the 3d and 4th course (until median 141.3 nmol/L, IQR 100.2-190.2). In parallel, folate levels increased most substantially after the first two HD-MTX courses (until median 401.6 nmol/L, IQR 163.3-594.2) after which levels plateaued during the 3d and 4th course (until median 411.5 nmol/L, IQR 240.3-665.6). The ratio folate/MTX-PG decreased significantly over time, which was mostly due to the relatively higher increase (delta) of MTX-PG. CONCLUSION: These results suggest that the increase in RBC folate levels does not seem to have a large effect on RBC MTX levels. Future studies, assessing competition of Leucovorin and MTX on other cellular mechanisms which might negatively affect treatment efficacy, are necessary.

Correction: Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia.

[This corrects the article DOI: 10.1371/journal.pone.0231588.].

Patient-derived oral mucosa organoids as an in vitro model for methotrexate induced toxicity in pediatric acute lymphoblastic leukemia.

We have recently established a protocol to grow wildtype human oral mucosa organoids. These three-dimensional structures can be maintained in culture long-term, do not require immortalization, and recapitulate the multilayered composition of the epithelial lining of the oral mucosa. Here, we validate the use of this model to study the effect of Leucovorin (LV) on Methotrexate (MTX)-induced toxicity. MTX is a chemotherapeutic agent used in the treatment of pediatric acute lymphoblastic leukemia. Although effective, the use of MTX often results in severe side-effects, including oral mucositis, which is characterized by epithelial cell death. Here, we show that organoids are sensitive to MTX, and that the addition of LV reduces MTX toxicity, in both a concentration- and timing-dependent manner. Additionally, we show that a 24 hour 'pretreatment' with LV reduces MTX-induced cell death, suggesting that such a pretreatment could decrease mucositis in patients. Taken together, we provide the first in vitro model to study the effect of MTX on wildtype oral mucosa cells. Our findings underscore the relevance of the clinically applied LV regimen and highlight the potential of this model to further optimize modifications in dosing and timing of Leucovorin on oral mucosa cells.

A multi-center assessment to compare residual allergenicity of partial hydrolyzed whey proteins in a murine model for cow's milk allergy - Comparison to the single parameter guinea pig model.

INTRODUCTION: This 4-center study is part of a project to validate a food allergy murine model for safety testing of hydrolyzed infant formulas. AIM: The aim of the current multi-center experiment was to evaluate the residual allergenicity of three partial hydrolyzed whey proteins (pWH) in a multiple-parameter cow's milk allergy murine model and to compare to the classically used guinea pig model. Previous work showed differences in the magnitude of the allergic response to whey between centers. To get a first insight in the effect of housing on the robustness of the mouse model, microbiota composition of non-sensitized mice was analyzed and compared between centers. METHODS: Mice were sensitized intragastrically (i.g.) with whey, pWH or eWH using cholera toxin as an adjuvant. In mice, whey-IgE/IgG1, acute allergic symptoms were determined upon whey challenge. Guinea pigs were orally sensitized ad libitum via the drinking water (day 0-37) and challenged intravenously with whey on day 49. The microbial composition in fecal samples was determined in non-sensitized mice in all 4 research centers before and after conduct of the study. RESULTS: Elevated levels of whey-IgG1 were detected in whey-sensitized mice in all centers. Except for pWH-A in center 4, we observed elevated levels of whey-IgE in whey-sensitized mice and mice sensitized with pWH-A, -B, -C. Center 2 was excluded from further analysis because of non-significant IgE levels in the positive control. In contrast to whey-mice, pWH-A treated mice showed no acute skin response, mMCP-1 release or change in body temperature upon whey challenge in all centers, which corresponds with the absence of anaphylactic shock symptoms in both the mouse and guinea pig model. pWH-B and -C induced anaphylactic shock symptoms in the guinea-pig and mice whereas results on the remaining allergic outcomes in mice were inconclusive. No differences in microbiota composition were measured in response to the challenge and Microbiota composition depended on the location of the centers. CONCLUSIONS: Both animal models showed comparable results on the residual allergenicity of partial hydrolyzed whey proteins, but none of the centers was able to differentiate between the residual sensitizing capacities of the pWH-B and -C based on a single elicitation parameter in the murine model. Differences in microbiota composition might contribute to the robustness of the food allergy murine model. For a well-balanced prediction on the potential allergenicity of hydrolyzed infant formulas a multiple murine parameter model is suggested to decrease the risk of false positive or false negative results. A future challenge is to develop an overall scoring system for proper risk assessment, taking all parameters into account.

Population Pharmacokinetics and Pharmacodynamics of Ciprofloxacin Prophylaxis in Pediatric Acute Lymphoblastic Leukemia Patients.

BACKGROUND: Ciprofloxacin is used as antimicrobial prophylaxis in pediatric acute lymphoblastic leukemia (ALL) to decrease infections with gram-negative bacteria. However, there are no clear guidelines concerning prophylactic dose. AIMS: To determine the pharmacokinetics and pharmacodynamics (PKPD) of ciprofloxacin prophylaxis in a pediatric ALL population. The effect of patient characteristics and antileukemic treatment on ciprofloxacin exposure, the area under the concentration time curve over minimal inhibitory concentration (AUC24/MIC) ratios, and emergence of resistance were studied. METHODS: A total of 615 samples from 129 children (0-18 years) with ALL were collected in a multicenter prospective study. A population pharmacokinetic model was developed. Microbiological cultures were collected prior to and during prophylaxis. An AUC24/MIC of ≥125 was defined as target ratio. RESULTS: A 1-compartment model with zero-order absorption and allometric scaling best described the data. No significant (P < .01) covariates remained after backward elimination and no effect of asparaginase or azoles were found. Ciprofloxacin AUC24 was 16.9 mg*h/L in the prednisone prophase versus 29.3 mg*h/L with concomitant chemotherapy. Overall, 100%, 81%, and 18% of patients at, respectively, MIC of 0.063, 0.125, and 0.25 mg/L achieved AUC24/MIC ≥ 125. In 13% of the patients, resistant bacteria were found during prophylactic treatment. CONCLUSION: Ciprofloxacin exposure shows an almost 2-fold change throughout the treatment of pediatric ALL. Depending on the appropriateness of 125 as target ratio, therapeutic drug monitoring or dose adjustments might be indicated for less susceptible bacteria starting from ≥ 0.125 mg/L to prevent the emergence of resistance and reach required targets for efficacy.

Neuroblastoma between 1990 and 2014 in the Netherlands: Increased incidence and improved survival of high-risk neuroblastoma.

PURPOSE: Long-term trends in neuroblastoma incidence and survival in unscreened populations are unknown. We explored trends in incidence, stage at diagnosis, treatment and survival of neuroblastoma in the Netherlands from 1990 to 2014. METHODS: The Netherlands Cancer Registry provided data on all patients aged <18 years diagnosed with a neuroblastoma. Trends in incidence and stage were evaluated by calculating the average annual percentage change (AAPC). Univariate and multivariable survival analyses were performed for stage 4 disease to test whether changes in treatment are associated with survival. RESULTS: Of the 593 newly diagnosed neuroblastoma cases, 45% was <18 months of age at diagnosis and 52% had stage 4 disease. The age-standardized incidence rate for stage 4 disease increased at all ages from 3.2 to 5.3 per million children per year (AAPC + 2.9%, p < .01). This increase was solely for patients ≥18 months old (3.0-5.4; AAPC +3.3%, p = .01). Five-year OS of all patients increased from 44 ± 5% to 61 ± 4% from 1990 to 2014 (p < .01) and from 19 ± 6% to 44 ± 6% (p < .01) for patients with stage 4 disease. Multivariable analysis revealed that high-dose chemotherapy followed by autologous stem cell rescue and anti-GD2-based immunotherapy were associated with this survival increase (HR 0.46, p < .01 and HR 0.37, p < .01, respectively). CONCLUSION: Incidence of stage 4 neuroblastoma increased exclusively in patients aged ≥18 months since 1990, whereas the incidence of other stages remained stable. The 5-year OS of stage 4 patients improved, mostly due to the introduction of high-dose chemotherapy followed by stem cell rescue and immunotherapy.

The effect of leucovorin rescue therapy on methotrexate-induced oral mucositis in the treatment of paediatric ALL: A systematic review.

INTRODUCTION: This study aimed to determine the efficacy of different Leucovorin regimens to reduce oral mucositis in children with acute lymphoblastic leukemia after high-dose Methotrexate (HD-MTX). METHODS: Twelve articles were included in a systematic literature review. Articles were categorized into low/medium/high risk of bias. RESULTS: As no randomized controlled trial assessing the effect of Leucovorin has been performed, the efficacy of Leucovorin to reduce oral mucositis remains unknown. Leucovorin was initiated at 24, 36 or 42 h after HD-MTX at a dose of 15 or 30 mg/m2. No meta-analysis could be performed as treatment regimens differed. When comparing studies with similar HD-MTX doses, we observed lower oral mucositis rates in regimens with higher cumulative doses of Leucovorin and early initiation of Leucovorin after MTX. CONCLUSION: Even though future studies are necessary, higher cumulative Leucovorin doses and early initiation of Leucovorin after start of MTX seem to reduce oral mucositis.

Lactulose synergizes with CpG-ODN to modulate epithelial and immune cells cross talk.

Lactulose, a non-digestible oligosaccharide and functional food, promotes Bifidobacteria growth. Here we show that lactulose, beyond its prebiotic action, may have direct immunomodulatory effects as well. In synergy with CpG-ODN, a bacterial DNA mimetic, lactulose enhances basolateral concentrations of IFN-γ, IL-10, and galectin-9 in the co-culture model of epithelial and immune cells.

'Why us?' Causal attributions of childhood cancer survivors, survivors' parents and community comparisons - a mixed methods analysis.

INTRODUCTION: Understanding the cause of their cancer is important for many cancer patients. Childhood cancer survivors'/survivors' parents' beliefs about cancer etiology are understudied. We aimed to assess survivors'/parents' beliefs about what causes childhood cancer, compared with beliefs in the community. We also investigated the influence of clinical and socio-demographic characteristics on the participants' beliefs about cancer etiology. METHODS: This two-stage study investigated the participants' beliefs, by using questionnaires assessing causal attributions related to childhood cancer (stage 1) and then undertaking telephone interviews (stage 2; survivors/survivors' parents only) to get an in-depth understanding of survivors'/survivors' parents beliefs. We computed multivariable regressions to identify factors associated with the most commonly endorsed attributions: bad luck/chance, environmental factors and genetics. We analyzed interviews using thematic analysis. RESULTS: Six hundred one individuals (64.6% survivors and 35.4% survivors' parents) and 510 community comparisons (53.1% community adults, 46.9% community parents) completed the question on causal attributions. We conducted 87 in-depth interviews. Survivors/survivors' parents (73.9%) were more likely to believe that chance/bad luck caused childhood cancer than community participants (42.4%). Community participants more frequently endorsed that genetics (75.3%) and environmental factors (65.3%) played a major role in childhood cancer etiology (versus survivors' and survivors' parents: genetics 20.6%, environmental factors: 19.3%). Community participants, participants with a first language other than English, and reporting a lower quality of life were less likely to attribute bad luck as a cause of childhood cancer. Community participants, all participants with a higher income and higher education were more likely to attribute childhood cancer etiology to environmental factors. CONCLUSION: Causal attributions differed between survivors/survivors' parents and community participants. Most of the parents and survivors seem to understand that there is nothing they have done to cause the cancer. Understanding survivors' and survivors' parents' causal attributions may be crucial to address misconceptions, offer access to services and to adapt current and future health behaviors.

A decrease in vitamin D levels is associated with methotrexate-induced oral mucositis in children with acute lymphoblastic leukemia.

PURPOSE: Children with acute lymphoblastic leukemia (ALL) are at increased risk of vitamin D deficiency, which might make them more susceptible to developing adverse events. Previous studies showed that low vitamin D levels were associated with an increased inflammatory mucosal state and impaired mucosal tissue barriers. We examined the prevalence of vitamin D deficiency and studied the association between vitamin D levels and methotrexate (MTX)-induced oral mucositis in pediatric ALL. METHODS: We assessed 25-hydroxyvitamin D (25(OH)D3) and 24,25-dihydroxyvitamin D (24,25(OH)2D3) levels in 99 children with ALL before the start of 4 × 5 g/m2 high-dose methotrexate (HD-MTX) (T0) and in 81/99 children after discontinuation of HD-MTX (T1). Two cutoff values for vitamin D deficiency exist: 25(OH)D3 levels < 30 and < 50 nmol/L. Oral mucositis was defined as grade ≥ 3 according to the National Cancer Institute Criteria. RESULTS: Vitamin D deficiency occurred in respectively 8% (< 30 nmol/L) and 33% (< 50 nmol/L) of the patients at T0, and more frequently in children > 4 years of age as compared to children between 1 and 4 years of age. A decrease in 25(OH)D3 levels during HD-MTX therapy was associated with developing severe oral mucositis (OR 1.6; 95% CI [1.1-2.4]). 25(OH)D3 and 24,25(OH)2D3 levels at T0 and the change in 24,25(OH)2D3 levels during therapy were not associated with the development of severe oral mucositis. CONCLUSIONS: This study showed that vitamin D deficiency occurs frequently in pediatric ALL patients above the age of 4 years. A decrease in 25(OH)D3 levels during MTX therapy was observed in children with ALL that developed severe oral mucositis.

Improved survival for children and young adolescents with acute myeloid leukemia: a Dutch study on incidence, survival and mortality.

Variation in survival of pediatric acute myeloid leukemia (pAML) over time and between Western European countries exists. The aim of the current study is to assess the progress made for the Dutch pAML population (0-17 years) during 1990-2015, based on trends in incidence, survival and mortality. Data from the population-based Netherlands Cancer Registry were merged with leukemia-related characteristics and treatment specifics from the Dutch Childhood Leukemia Study Group (Dutch Childhood Oncology Group (DCOG) from 2002 onwards). Mortality data (1980-2016) were obtained from the cause of death registry of Statistics Netherlands. Trend analyses were performed over time and by treatment protocol. Between 1990 and 2015, a total of 635 children aged 0-17 years were diagnosed with AML for an average of 25 patients (range 18-36) per year. There was a slight increase in the incidence at age 1-4 years (average annual percentage change (AAPC) of +2.2% per year (95% CI 0.8-3.5, p < 0.01)). Overall, the 5-year survival significantly improved over the past 26 years and nearly doubled from 40% in the early 1990s to 74% in 2010-2015. Multivariable analysis showed a 49% reduction in risk of death for pAML patients treated according to the latest DB-AML 01 protocol (p = 0.03). The continuing decrease of mortality (AAPC -2.8% per year (95% CI -4.1 to -1.5)) supports the conclusion of true progress against pAML in the Netherlands.

Topically used corticosteroids: What is the big picture of drug product degradation?

Corticosteroids are widely used in topical formulations such as creams (aqueous) and ointments (non-aqueous). The generally used corticosteroids show large molecular resemblance, where especially the 20-keto-21-hydroxyl group bound to the 17 carbon is important for their chemical stability. Oxidation in both aqueous and non-aqueous environment occurs for triamcinolone acetonide (TCA), hydrocortisone (HC) and desoximethasone (DS). Besides the 20-keto-21-hydroxyl group, TCA, HC and DS have different other moieties attached to the same C17. These moieties are shown to influence not only the type of degradation product formed but also the degradation kinetics. Seven degradation products are found in total and a degradation mechanism is proposed. Furthermore the transesterfication of betamethasone-17-valerate to betamethasone-21-valerate is shown to occur both in aqueous and non-aqueous environment. Finally, a comprehensive scheme of degradation pathways is presented that is applicable for both aqueous and non-aqueous formulations.

MN1 overexpression is driven by loss of DNMT3B methylation activity in inv(16) pediatric AML.

In acute myeloid leukemia (AML), specific genomic aberrations induce aberrant methylation, thus directly influencing the transcriptional programing of leukemic cells. Therefore, therapies targeting epigenetic processes are advocated as a promising therapeutic tool for AML treatment. However, to develop new therapies, a comprehensive understanding of the mechanism(s) driving the epigenetic changes as a result of acquired genetic abnormalities is necessary. This understanding is still lacking. In this study, we performed genome-wide CpG-island methylation profiling on pediatric AML samples. Six differentially methylated genomic regions within two genes, discriminating inv(16)(p13;q22) from non-inv(16) pediatric AML samples, were identified. All six regions had a hypomethylated phenotype in inv(16) AML samples, and this was most prominent at the regions encompassing the meningioma (disrupted in balanced translocation) 1 (MN1) oncogene. MN1 expression primarily correlated with the methylation level of the 3' end of the MN1 exon-1 locus. Decitabine treatment of different cell lines showed that induced loss of methylation at the MN1 locus can result in an increase of MN1 expression, indicating that MN1 expression is coregulated by DNA methylation. To investigate this methylation-associated mechanism, we determined the expression of DNA methyltransferases in inv(16) AML. We found that DNMT3B expression was significantly lower in inv(16) samples. Furthermore, DNMT3B expression correlated negatively with MN1 expression in pediatric AML samples. Importantly, depletion of DNMT3B impaired remethylation efficiency of the MN1 exon-1 locus in AML cells after decitabine exposure. These findings identify DNMT3B as an important coregulator of MN1 methylation. Taken together, this study shows that the methylation level of the MN1 exon-1 locus regulates MN1 expression levels in inv(16) pediatric AML. This methylation level is dependent on DNMT3B, thus suggesting a role for DNMT3B in leukemogenesis in inv(16) AML, through MN1 methylation regulation.

RAS pathway mutations as a predictive biomarker for treatment adaptation in pediatric B-cell precursor acute lymphoblastic leukemia.

RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged, BCR-ABL1-like and B-other cases (50-70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1- and BCR-ABL1-rearranged cases (27-4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo. Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse, most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations may designate patients eligible for MEK inhibitor treatment.

The HDAC inhibitor panobinostat (LBH589) exerts in vivo anti-leukaemic activity against MLL-rearranged acute lymphoblastic leukaemia and involves the RNF20/RNF40/WAC-H2B ubiquitination axis.

MLL-rearranged acute lymphoblastic leukaemia (ALL) represents an aggressive malignancy in infants (<1 year of age), associated with poor outcome. Current treatment intensification is not further possible, and novel therapy strategies are needed. Notably, MLL-rearranged ALL is characterised by a strongly deregulated epigenome and shows sensitivity to epigenetic perturbators. Here we demonstrate the in vivo efficacy of the histone deacetylase inhibitor panobinostat (LBH589) using xenograft mouse models of MLL-rearranged ALL. Panobinostat monotherapy showed strong anti-leukaemic effects, extending survival and reducing overall disease burden. Comprehensive molecular analyses in vitro showed that this anti-leukaemic activity involves depletion of H2B ubiquitination via suppression of the RNF20/RNF40/WAC E3 ligase complex; a pivotal pathway for MLL-rearranged leukaemic maintenance. Knockdown of WAC phenocopied loss of H2B ubiquitination and concomitant cell death induction. These combined data demonstrate that panobinostat cross-inhibits multiple epigenetic pathways, ultimately contributing to its highly efficacious targeting of MLL-rearranged ALL.