Exhaled nitric oxide is associated with inflammatory biomarkers and risk of acute respiratory exacerbations in children with HIV-associated chronic lung disease.

OBJECTIVES: Chronic lung disease is a recognized complication in children with HIV. Acute respiratory exacerbations (ARE) are common among this group and cause significant morbidity. Exhaled nitric oxide (eNO) is a known marker of local airway inflammation. We investigated the association between eNO and ARE, biomarkers of systemic inflammation, and the effect of azithromycin on eNO levels. METHODS: Individuals aged 6-19 years with HIV-associated chronic lung disease in Harare, Zimbabwe, were enrolled in a placebo-controlled randomized trial investigating the effect of 48-week azithromycin treatment on lung function and ARE. eNO levels and biomarkers were measured at inclusion and after treatment in a consecutively enrolled subset of participants. Linear regression and generalized linear models were used to study associations between eNO and ARE, biomarkers, and the effect of azithromycin on eNO levels. RESULTS: In total, 172 participants were included in this sub-study, 86 from the placebo group and 86 from the azithromycin group. Participants experiencing at least one ARE during follow-up had significantly higher eNO levels at baseline than participants who did not (geometric mean ratio 1.13, 95% confidence interval [CI] 1.03-1.24, p = 0.015), adjusted for trial arm, age, sex and history of tuberculosis. Matrix metalloproteinase (MMP)-3, -7, and -10 were significantly associated with higher baseline eNO levels. At 48 weeks, azithromycin treatment did not affect eNO levels (geometric mean ratio 0.86, 95% CI 0.72-1.03, p = 0.103). CONCLUSION: Higher baseline eNO levels were a risk factor for ARE. eNO was associated with proinflammatory biomarkers previously found to contribute to the development of chronic lung disease. The potential use of eNO as a marker of inflammation and risk factor for ARE in HIV-associated chronic lung disease needs further investigation.

A Nationwide Study of GATA2 Deficiency in Norway-the Majority of Patients Have Undergone Allo-HSCT.

PURPOSE: GATA2 deficiency is a rare primary immunodeficiency that has become increasingly recognized due to improved molecular diagnostics and clinical awareness. The only cure for GATA2 deficiency is allogeneic hematopoietic stem cell transplantation (allo-HSCT). The inconsistency of genotype-phenotype correlations makes the decision regarding "who and when" to transplant challenging. Despite considerable morbidity and mortality, the reported proportion of patients with GATA2 deficiency that has undergone allo-HSCT is low (~ 35%). The purpose of this study was to explore if detailed clinical, genetic, and bone marrow characteristics could predict end-point outcome, i.e., death and allo-HSCT. METHODS: All medical genetics departments in Norway were contacted to identify GATA2 deficient individuals. Clinical information, genetic variants, treatment, and outcome were subsequently retrieved from the patients' medical records. RESULTS: Between 2013 and 2020, we identified 10 index cases or probands, four additional symptomatic patients, and no asymptomatic patients with germline GATA2 variants. These patients had a diverse clinical phenotype dominated by cytopenia (13/14), myeloid neoplasia (10/14), warts (8/14), and hearing loss (7/14). No valid genotype-phenotype correlations were found in our data set, and the phenotypes varied also within families. We found that 11/14 patients (79%), with known GATA2 deficiency, had already undergone allo-HSCT. In addition, one patient is awaiting allo-HSCT. The indications to perform allo-HSCT were myeloid neoplasia, disseminated viral infection, severe obliterating bronchiolitis, and/or HPV-associated in situ carcinoma. Two patients died, 8 months and 7 years after allo-HSCT, respectively. CONCLUSION: Our main conclusion is that the majority of patients with symptomatic GATA2 deficiency will need allo-HSCT, and a close surveillance of these patients is important to find the "optimal window" for allo-HSCT. We advocate a more offensive approach to allo-HSCT than previously described.

Detection mode of childhood acute lymphoblastic leukaemia relapse and its effect on survival: a Nordic population-based cohort study.

Relapse constitutes the greatest threat to event-free survival after completion of treatment for childhood acute lymphoblastic leukaemia (ALL). However, evidence on optimal follow-up schedules is limited. The aims of the present population-based cohort study were to assess the value of current follow-up schedules after completion of Nordic Society of Paediatric Haematology and Oncology ALL protocol treatment and to estimate the impact of relapse detection mode on overall survival (OS). Among 3262 patients diagnosed between 1992 and 2014 and who completed treatment, 338 developed a relapse. Relapse detection was equally distributed between extra visits (50·8%) and scheduled follow-up visits (49·2%). All cases detected at an extra visit and 64·3% of cases detected at a scheduled visit presented with symptoms or objective findings. Neither the mode of detection {adjusted hazard ratio 0·95, [95% confidence interval (CI) 0·61-1·48] for scheduled visits} nor the duration of symptoms was an independent risk factor for OS after relapse. The estimated number of scheduled blood samples needed to diagnose one subclinical relapse during the first 5 years after treatment cessation was 1269 (95% CI 902-1637). In conclusion, based on OS data, scheduled visits after cessation of therapy seem to yield no extra benefit. These results should frame future follow-up strategies.

Chronic iron deficiency and anaemia were highly prevalent in a population-based longitudinal study among adolescent girls.

AIM: The combination of iron deficiency and anaemia is a major health problem, and adolescents are an at-risk group. The main aim of this study was to explore the magnitude of these conditions among adolescents aged 15-19 and identify possible associated risk factors. METHODS: This population-based longitudinal study of adolescents in North Norway was conducted in 2010-2011, with a follow-up two years later. Repeated measurements of iron deficiency and anaemia and its possible risk factors were studied in 309 girls and 273 boys. RESULTS: Iron deficiency and anaemia were found in 18.1% and 19.9% of girls and 1.6% and 2.9% of boys in the first study and about half of the cases were chronic two years later. Most girls had moderate iron deficiency (14.5%) and mild anaemia (16.0%). Daily milk consumption was associated with increased iron deficiency in girls (odds ratio 2.3, 95% confidence interval 1.1-4.9), and the most physically active girls had the lowest levels of iron deficiency (odds ratio 0.4, 95% confidence intervaI 0.2-0.9). Iron deficiency was the most important risk factor for chronic anaemia in girls. CONCLUSION: The results of this study highlight the importance of iron deficiency screening and treatment for adolescent girls.

Composition of Gut Microbiota of Children and Adolescents With Perinatal Human Immunodeficiency Virus Infection Taking Antiretroviral Therapy in Zimbabwe.

BACKGROUND: Human immunodeficiency virus (HIV) infection causes impairment of the gastrointestinal barrier, with substantial depletion of CD4+ T cells in the gut. Antiretroviral therapy (ART) restores CD4+ counts and may have beneficial effects on gut microbiota in adults. Little is known about effect of long-term ART on gut microbiome in HIV-infected children. We investigated composition of gut microbiota in HIV-infected and -uninfected children and assessed associations between gut microbiota and patient characteristics. METHODS: In a cross-sectional study, rectal swabs were collected from 177 HIV-infected and 103 HIV-uninfected controls. Gut microbial composition was explored using 16S ribosomal ribonucleic acid sequencing. RESULTS: Human immunodeficiency virus-infected children had significantly lower alpha-diversity and higher beta-diversity compared to HIV-uninfected. No association was observed between microbiome diversity and CD4+ T-cell count, HIV viral load, or HIV-associated chronic lung disease. We found enriched levels of Corynebacterium (P < .01), Finegoldia (P < .01), and Anaerococcus (P < .01) in HIV-infected participants and enrichment of Enterobacteriaceae (P = .02) in participants with low CD4+ counts (<400 cells/mm3). Prolonged ART-treatment (≥10 years) was significantly associated with a richer gut microbiota by alpha diversity. CONCLUSIONS: Human immunodeficiency virus-infected children have altered gut microbiota. Prolonged ART may restore the richness of the microbiota closer to that of HIV-uninfected children.

No additional long-term effect of group vs individual family intervention in the treatment of childhood obesity-A randomised trial.

AIM: Long-term evaluations of childhood obesity treatments are needed. We examined changes in weight and cardiometabolic risk 1 year after children completed individual family or group-based weight management interventions. METHODS: In 2009-2010, 6- to 12-year-old children with overweight or obesity from Finnmark and Troms (Norway) were recruited after media coverage and randomised to 24 months of individual family (n = 49) or group intervention (n = 48). Individual family intervention included counselling by a paediatric hospital team and a public health nurse in the local community. Group intervention included meetings with other families and a multidisciplinary hospital team, weekly physical activity sessions and a family camp. The primary outcome body mass index (BMI) and cardiometabolic risk factors were analysed 12 months after intervention. RESULTS: From baseline to 36 months, children's BMI increased 3.0 kg/m2 in individual family and 2.1 kg/m2 in group intervention (between-group -0.9kg/m2 , P = 0.096). Data were available from 62 children (64%). Between-group differences in C peptide (P = 0.01) were detected in favour of group intervention. Pooled data from both treatment groups showed continued decrease in BMI standard deviation score (P < 0.001). CONCLUSION: No between-group difference in BMI was observed 12 months after intervention. Both groups combined showed sustained decrease in BMI standard deviation score.

An epidemic of meningococcal disease in children in North Norway in the 1970s and 1980s was dominated by a hypervirulent group B strain.

AIM: We examined children hospitalised for invasive meningococcal disease, a leading cause of paediatric sepsis, in Troms County, North Norway, from 1973 to 2016, including the epidemic in the 1970s and 1980s. METHODS: This study was a retrospective review of children under the age of 15 years who were hospitalised for meningococcal disease at the University Hospital of North Norway and Harstad Hospital. We studied hospital and bacteriological records to determine the incidence rates and phenotypes involved. RESULTS: There were 300 cases under 15 years and an incidence rate of 17 per 100,000 cases for 1973-2016. This was broken down into the following: 1973-1980 (n = 130, 49), 1981-1990 (n = 129, 39), and 1991-2016 (n = 41, 4.7), respectively. There were 21 (7%) deaths. Phenotype B:15:P1.7,16 was more common than the other phenotypes in the epidemic period before 1990 than after 1990 (p = 0.02) and had a significantly lower mortality rate than the other phenotypes (p = 0.04). Later years showed a more heterogenous phenotype distribution. Serogroup B was the dominant serogroup. CONCLUSION: The B:15:P1.7,6 strain was more prevalent during the Norwegian epidemic of invasive meningococcal disease, but had a significantly lower mortality rate. The phenotype distribution was more heterogeneous after 1990. The dominant serogroup was B.

Treatment of two cases on the same day of intrathecal methotrexate overdose using cerebrospinal fluid exchange and intrathecal instillation of carboxypeptidase-G2.

BACKGROUND: Two 14-year old boys with acute lymphocytic leukemia were treated according to the NOPHO-ALL-08 protocol with intrathecal methotrexate (MTX) on the same day. Due to a preparation error in the hospital pharmacy, they were both given 240 mg of MTX instead of the prescribed 12 mg. Treatment (or methods): Both patients developed acute neurotoxicity with confusion, pain and seizures. Intravenous dexamethasone and folinic acid (leucovorin) was given. Exchange of cerebrospinal fluid was performed. Intrathecal glucarpidase (carboxypeptidase-G2) was administered after 11 h. RESULTS: One patient developed a toxic arachnoiditis. Three years after the incident, one patient has no neurological or neuropsychological sequelae after the overdose, while the other reports some loss of short-term memory. CONCLUSION: Fast recognition and treatment of intrathecal MTX overdose is critical to survival and outcome. Efforts to prevent such overdoses are of vital importance.

Whole-genome sequencing reveals clonal expansion of multiresistant Staphylococcus haemolyticus in European hospitals.

OBJECTIVES: Staphylococcus haemolyticus is an emerging cause of nosocomial infections, primarily affecting immunocompromised patients. A comparative genomic analysis was performed on clinical S. haemolyticus isolates to investigate their genetic relationship and explore the coding sequences with respect to antimicrobial resistance determinants and putative hospital adaptation. METHODS: Whole-genome sequencing was performed on 134 isolates of S. haemolyticus from geographically diverse origins (Belgium, 2; Germany, 10; Japan, 13; Norway, 54; Spain, 2; Switzerland, 43; UK, 9; USA, 1). Each genome was individually assembled. Protein coding sequences (CDSs) were predicted and homologous genes were categorized into three types: Type I, core genes, homologues present in all strains; Type II, unique core genes, homologues shared by only a subgroup of strains; and Type III, unique genes, strain-specific CDSs. The phylogenetic relationship between the isolates was built from variable sites in the form of single nucleotide polymorphisms (SNPs) in the core genome and used to construct a maximum likelihood phylogeny. RESULTS: SNPs in the genome core regions divided the isolates into one major group of 126 isolates and one minor group of isolates with highly diverse genomes. The major group was further subdivided into seven clades (A-G), of which four (A-D) encompassed isolates only from Europe. Antimicrobial multiresistance was observed in 77.7% of the collection. High levels of homologous recombination were detected in genes involved in adherence, staphylococcal host adaptation and bacterial cell communication. CONCLUSIONS: The presence of several successful and highly resistant clones underlines the adaptive potential of this opportunistic pathogen.

Efficacy of a synthetic antimicrobial peptidomimetic versus vancomycin in a Staphylococcus epidermidis device-related murine peritonitis model.

OBJECTIVES: Biofilm-forming Staphylococcus epidermidis is a prevalent cause of peritonitis during peritoneal dialysis. We compared the efficacy of a synthetic antimicrobial peptidomimetic (Ltx21) versus vancomycin in a murine model mimicking a device-related peritonitis. METHODS: Silicone implants, pre-colonized with an S. epidermidis biofilm, were inserted into the peritoneal cavity of BALB/c mice. Three groups (36 mice in each) with pre-colonized implants received intraperitoneal treatment with Ltx21, vancomycin or placebo. Mice were euthanized on day 3 (n = 12), day 6 (n = 12) or day 8 (n = 12) post-implantation. Controls were mice with sterile implants (n = 18) and mice without surgery (n = 6). Bacterial reductions in cfu were analysed from implants and peritoneal fluid (PF). Inflammatory responses in serum and PF were measured. RESULTS: Vancomycin resulted in a stronger reduction in cfu counts, both on pre-colonized implants and in PF, compared with Ltx21 and placebo. Complete bacterial clearance of the implants was not achieved in any of the groups. The implants pre-colonized with S. epidermidis 1457 resulted in a low-grade peritonitis. We observed, only on day 6, a significant increase in the PF leucocyte count in the group with pre-colonized implants compared with the group with sterile implants (P = 0.0364). CONCLUSIONS: Treatment with vancomycin or Ltx21 was not sufficient to achieve complete bacterial clearance of implants, underlining the difficulties of treating such infections. The low-grade infection may attenuate the inflammatory response and contribute to impaired bacterial clearance.

Staphylococcus epidermidis biofilms induce lower complement activation in neonates as compared with adults.

BACKGROUND: Staphylococcus epidermidis (SE) is an important cause of late-onset sepsis in neonates. SE frequently produces a polysaccharide intercellular adhesin (PIA) biofilm, important in the pathogenesis of these infections. Little is known about how the neonatal innate immune system reacts to SE biofilm-associated infections. Our hypothesis was that SE biofilms induce a lower complement activation in neonates as compared with adults. METHODS: Cord blood from term infants (n = 15) and blood from adults (n = 6) were studied in an ex vivo whole-blood sepsis model. A PIA biofilm-producing strain (SE1457) and its isogenic mutant (M10), producing a non-PIA biofilm, were used. RESULTS: Both SE biofilms induced stronger complement activation in adult than in cord blood (P ≤ 0.033). We found lower levels of antibodies toward both PIA (P = 0.002) and the whole bacterium (P = 0.001) in cord vs. adult blood. By contrast, the interleukin-8 (IL-8) and IL-6 secretion were higher in cord than in adult blood (P ≤ 0.002). The PIA biofilm induced stronger complement activation than the non-PIA biofilm. CONCLUSION: We conclude that the neonatal complement system exhibits a maturational deficiency. This may reduce the ability of neonates to combat biofilm-associated SE infections.

Hsa-miR-323a-3p functions as a tumor suppressor and targets STAT3 in neuroblastoma cells.

Background: Studies conducted in the last decades have revealed a role for the non-coding microRNAs (miRNAs) in cancer development and progression. Several miRNAs within the chromosome region 14q32, a region commonly deleted in cancers, are associated with poor clinical outcome in the childhood cancer neuroblastoma. We have previously identified miR-323a-3p from this region to be downregulated in chemotherapy treated neuroblastoma cells compared to pre-treatment cells from the same patients. Furthermore, in neuroblastoma tumors, this miRNA is downregulated in advanced stage 4 disease compared to stage 1-2. In this study, we attempt to delineate the unknown functional roles of miR-323a-3p in neuroblastoma. Methods: Synthetic miRNA mimics were used to overexpress miR-323a-3p in neuroblastoma cell lines. To investigate the functional roles of miR-323a-3p, cell viability assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction, luciferase reporter assay and western blot were conducted on the neuroblastoma cell lines Kelly, SH-SY5Y and SK-N-BE(2)-C. Results: Ectopic expression of miR-323a-3p resulted in marked reduction of cell viability in Kelly, SH-SY5Y and SK-N-BE(2)-C by causing G1-cell cycle arrest in Kelly and SH-SY5Y and apoptosis in all the cell lines tested. Furthermore, mRNA and protein levels of signal transducer and activator of transcription 3 (STAT3) were reduced upon miR-323a-3p overexpression. A direct binding of the miR-323a-3p to the 3'UTR of STAT3 was experimentally validated by luciferase reporter assay, where miR-323a-3p reduced luminescent signal from full length STAT3 3'UTR luciferase reporter, but not from a reporter with mutation in the predicted seed sequence. Conclusions: miR-323a-3p inhibits growth of neuroblastoma cell lines through G1-cell cycle arrest and apoptosis, and the well-known oncogene STAT3 is a direct target of this miRNA.

Multimodal classification of molecular subtypes in pediatric acute lymphoblastic leukemia.

Genomic analyses have redefined the molecular subgrouping of pediatric acute lymphoblastic leukemia (ALL). Molecular subgroups guide risk-stratification and targeted therapies, but outcomes of recently identified subtypes are often unclear, owing to limited cases with comprehensive profiling and cross-protocol studies. We developed a machine learning tool (ALLIUM) for the molecular subclassification of ALL in retrospective cohorts as well as for up-front diagnostics. ALLIUM uses DNA methylation and gene expression data from 1131 Nordic ALL patients to predict 17 ALL subtypes with high accuracy. ALLIUM was used to revise and verify the molecular subtype of 281 B-cell precursor ALL (BCP-ALL) cases with previously undefined molecular phenotype, resulting in a single revised subtype for 81.5% of these cases. Our study shows the power of combining DNA methylation and gene expression data for resolving ALL subtypes and provides a comprehensive population-based retrospective cohort study of molecular subtype frequencies in the Nordic countries.

DNA methylation for subtype classification and prediction of treatment outcome in patients with childhood acute lymphoblastic leukemia.

Despite improvements in the prognosis of childhood acute lymphoblastic leukemia (ALL), subgroups of patients would benefit from alternative treatment approaches. Our aim was to identify genes with DNA methylation profiles that could identify such groups. We determined the methylation levels of 1320 CpG sites in regulatory regions of 416 genes in cells from 401 children diagnosed with ALL. Hierarchical clustering of 300 CpG sites distinguished between T-lineage ALL and B-cell precursor (BCP) ALL and between the main cytogenetic subtypes of BCP ALL. It also stratified patients with high hyperdiploidy and t(12;21) ALL into 2 subgroups with different probability of relapse. By using supervised learning, we constructed multivariate classifiers by external cross-validation procedures. We identified 40 genes that consistently contributed to accurate discrimination between the main subtypes of BCP ALL and gene sets that discriminated between subtypes of ALL and between ALL and controls in pairwise classification analyses. We also identified 20 individual genes with DNA methylation levels that predicted relapse of leukemia. Thus, methylation analysis should be explored as a method to improve stratification of ALL patients. The genes highlighted in our study are not enriched to specific pathways, but the gene expression levels are inversely correlated to the methylation levels.

High prevalence of overweight and obesity among 6-year-old children in Finnmark County, North Norway.

AIM: The aim was to determine the prevalence of overweight and obesity among 6-year-old children in Finnmark, the northernmost county of Norway. METHODS: This is a survey of 1774 children born during 1999 and 2000 from 18 of 19 child healthcare centres in Finnmark. Body mass index data extracted retrospectively in 2007 from health records at the age of 6 years were compared with international definitions of over- and underweight. The prevalence figures were further compared with socio-demographic figures on municipality level. RESULTS: Overall, 19% of the children were classified as overweight or obese; 5% were classified as obese. The prevalence of overweight and obesity was higher among girls (22%) than among boys (16%) (p < 0.01). The prevalence of underweight was 8% among both girls and boys. Despite large variations in the prevalence of overweight and obesity between municipalities (9-35%), no association was found with municipality figures on socio-demographic factors. CONCLUSION: In the northernmost county Finnmark, the prevalence of overweight including obesity among 6-year-old children was somewhat higher than in previous surveys from Norway, especially among girls.

Inhibition of mir-21, which is up-regulated during MYCN knockdown-mediated differentiation, does not prevent differentiation of neuroblastoma cells.

BACKGROUND: Neuroblastoma is a malignant childhood tumour arising from precursor cells of the sympathetic nervous system. Genomic amplification of the MYCN oncogene is associated with dismal prognosis. For this group of high-risk tumours, the induction of tumour cell differentiation is part of current treatment protocols. MicroRNAs (miRNAs) are small non-coding RNA molecules that effectively reduce the translation of target mRNAs. MiRNAs play an important role in cell proliferation, apoptosis, differentiation and cancer. In this study, we investigated the role of N-myc on miRNA expression in MYCN-amplified neuroblastoma. We performed a miRNA profiling study on SK-N-BE (2) cells, and determined differentially expressed miRNAs during differentiation initiated by MYCN knockdown, using anti-MYCN short-hairpin RNA (shRNA) technology. RESULTS: Microarray analyses revealed 23 miRNAs differentially expressed during the MYCN knockdown-mediated neuronal differentiation of MNA neuroblastoma cells. The expression changes were bidirectional, with 11 and 12 miRNAs being up- and down-regulated, respectively. Among the down-regulated miRNAs, we found several members of the mir-17 family of miRNAs. Mir-21, an established oncomir in a variety of cancer types, became strongly up-regulated upon MYCN knockdown and the subsequent differentiation. Neither overexpression of mir-21 in the high-MYCN neuroblastoma cells, nor repression of increased mir-21 levels during MYCN knockdown-mediated differentiation had any significant effects on cell differentiation or proliferation. CONCLUSIONS: We describe a subset of miRNAs that were altered during the N-myc deprived differentiation of MYCN-amplified neuroblastoma cells. In this context, N-myc acts as both an activator and suppressor of miRNA expression. Mir-21 was up-regulated during cell differentiation, but inhibition of mir-21 did not prevent this process. We were unable to establish a role for this miRNA during differentiation and proliferation of the two neuroblastoma cell lines used in this study.

Temporal changes in incidence of relapse and outcome after relapse of childhood acute lymphoblastic leukemia over three decades; a Nordic population-based cohort study.

Relapse remains the main obstacle to curing childhood acute lymphoblastic leukemia (ALL). The aims of this study were to compare incidence of relapse, prognostic factors, and survival after relapse between three consecutive Nordic Society of Pediatric Hematology and Oncology trials. Relapse occurred as a primary event in 638 of 4 458 children (1.0-14.9 years) diagnosed with Ph-negative ALL between 1992 and 2018. The 5-year cumulative incidence of relapse was 17.3% (95% CI 15.4-19.2%) and 16.5% (95% CI 14.3-18.8%) for patients in the ALL1992 and ALL2000 trials, respectively, but decreased to 8.4% (95% CI 7.0-10.1%) for patients in the ALL2008 trial. No changes in duration of first complete remission and site of relapse were observed over time; however, high hyperdiploidy, and t(12;21) decreased in the ALL2008 trial. The 4-year overall survival after relapse was 56.6% (95% CI 52.5-60.5%) and no statistically significant temporal improvements were observed. Age ≥10 years, T-cell immunophenotype, bone-marrow involvement, early and very early relapse, hypodiploidy, and Down syndrome all independently predicted worse outcome after relapse. Improvements in the primary treatment of childhood ALL has resulted in fewer relapses. However, failure to improve outcome of remaining relapses suggests a selection of harder-to-cure relapses and calls for new therapeutic strategies.

The Norwegian childhood cancer biobank.

BACKGROUND: The rapidly expanding era of "omics" research is highly dependent on the availability of quality-proven biological material, especially for rare conditions such as pediatric malignancies. Professional biobanks provide such material, focusing on standardized collection and handling procedures, distinctive quality measurements, traceability of storage conditions, and accessibility. For pediatric malignancies, traditional tumor biobanking is challenging due to the rareness and limited amount of tissue and blood samples. The higher molecular heterogeneity, lower mutation rates, and unique genomic landscapes, however, renders biobanking of this tissue even more crucial. AIM: The aim of this study was to test and establish methods for a prospective and centralized biobank for infants, children, and adolescents up to 18 years of age diagnosed with cancer in Norway. METHODS: Obtain judicial and ethical approvals and administration through a consortium, steering committee, and advisory board. Develop pipelines including SOPs for all aspects in the biobank process, including collection, processing and storing of samples and data, as well of quality controlling, safeguarding, distributing, and transport. RESULTS: The childhood cancer biobanking started at Oslo University Hospital in March 2017 and was from 2019 run as a national Norwegian Childhood Cancer Biobank. Informed consent and biological samples are collected regionally and stored centrally. Approximately 12 000 samples from 510 patients and have been included by January 1, 2021, representing a 96% consent and participation rate among our newly diagnosed patients. CONCLUSION: A well-functioning nationwide collection and centralized biobank with standardized procedures and national storage for pediatric malignancies has been established with a high acceptance among families.

MYCN-regulated miRNA-92 inhibits secretion of the tumor suppressor DICKKOPF-3 (DKK3) in neuroblastoma.

The MYCN oncogene is frequently amplified in neuroblastoma. It is one of the most consistent markers of bad prognosis for this disease. Dickkopf-3 (DKK3) is a secreted protein of the DKK family of Wnt regulators. It functions as a tumor suppressor in a range of cancers, including neuroblastoma. MYCN was recently found to downregulate DKK3 mRNA. In this study, we show that MYCN knockdown in MYCN-amplified (MNA) neuroblastoma cell lines increases secretion of endogenous DKK3 to the culture media. MicroRNAs (miRNAs) are ∼20 nt long single-stranded RNA molecules that downregulate messenger RNAs by targeting the 3' untranslated region (3'UTR). Many miRNAs regulate genes involved in the pathogenesis of cancer and are extensively deregulated in different tumors. Using miRNA target prediction software, we found several MYCN-regulated miRNAs that could target the 3'UTR sequence of DKK3, including mir-92a, mir-92b and let-7e. Luciferase expression from a reporter vector containing the DKK3-3'UTR was decreased when this construct was cotransfected with mir-92a, mir-92b or let-7e in HEK293 cells. Mutation of the mir-92 seed sequence in the 3'UTR completely rescued the observed decrease in reporter expression when cotransfected with mir-92a and mir-92b. Antagomir and miRNA-mimic transfections in neuroblastoma cell lines confirmed that DKK3 secretion to the culture media is regulated by mir-92. Consistent with reports from other cancers, we found DKK3 to be expressed in the endothelium of primary neuroblastoma samples and to be absent in tumors with MYCN amplification. Our data demonstrate that MYCN-regulated miRNAs are able to modulate the expression of the tumor suppressor DKK3 in neuroblastoma.