Signalling input from divergent pathways subverts B cell transformation.

Malignant transformation of cells typically involves several genetic lesions, whose combined activity gives rise to cancer1. Here we analyse 1,148 patient-derived B-cell leukaemia (B-ALL) samples, and find that individual mutations do not promote leukaemogenesis unless they converge on one single oncogenic pathway that is characteristic of the differentiation stage of transformed B cells. Mutations that are not aligned with this central oncogenic driver activate divergent pathways and subvert transformation. Oncogenic lesions in B-ALL frequently mimic signalling through cytokine receptors at the pro-B-cell stage (via activation of the signal-transduction protein STAT5)2-4 or pre-B-cell receptors in more mature cells (via activation of the protein kinase ERK)5-8. STAT5- and ERK-activating lesions are found frequently, but occur together in only around 3% of cases (P = 2.2 × 10-16). Single-cell mutation and phospho-protein analyses reveal the segregation of oncogenic STAT5 and ERK activation to competing clones. STAT5 and ERK engage opposing biochemical and transcriptional programs that are orchestrated by the transcription factors MYC and BCL6, respectively. Genetic reactivation of the divergent (suppressed) pathway comes at the expense of the principal oncogenic driver and reverses transformation. Conversely, deletion of divergent pathway components accelerates leukaemogenesis. Thus, persistence of divergent signalling pathways represents a powerful barrier to transformation, while convergence on one principal driver defines a central event in leukaemia initiation. Pharmacological reactivation of suppressed divergent circuits synergizes strongly with inhibition of the principal oncogenic driver. Hence, reactivation of divergent pathways can be leveraged as a previously unrecognized strategy to enhance treatment responses.

Characterization of the innate immune response to Streptococcus pneumoniae infection in zebrafish.

Streptococcus pneumoniae (pneumococcus) is one of the most frequent causes of pneumonia, sepsis and meningitis in humans, and an important cause of mortality among children and the elderly. We have previously reported the suitability of the zebrafish (Danio rerio) larval model for the study of the host-pathogen interactions in pneumococcal infection. In the present study, we characterized the zebrafish innate immune response to pneumococcus in detail through a whole-genome level transcriptome analysis and revealed a well-conserved response to this human pathogen in challenged larvae. In addition, to gain understanding of the genetic factors associated with the increased risk for severe pneumococcal infection in humans, we carried out a medium-scale forward genetic screen in zebrafish. In the screen, we identified a mutant fish line which showed compromised resistance to pneumococcus in the septic larval infection model. The transcriptome analysis of the mutant zebrafish larvae revealed deficient expression of a gene homologous for human C-reactive protein (CRP). Furthermore, knockout of one of the six zebrafish crp genes by CRISPR-Cas9 mutagenesis predisposed zebrafish larvae to a more severe pneumococcal infection, and the phenotype was further augmented by concomitant knockdown of a gene for another Crp isoform. This suggests a conserved function of C-reactive protein in anti-pneumococcal immunity in zebrafish. Altogether, this study highlights the similarity of the host response to pneumococcus in zebrafish and humans, gives evidence of the conserved role of C-reactive protein in the defense against pneumococcus, and suggests novel host genes associated with pneumococcal infection.

The clinical picture of ERCC6L2 disease: from bone marrow failure to acute leukemia.

Biallelic germ line excision repair cross-complementing 6 like 2 (ERCC6L2) variants strongly predispose to bone marrow failure (BMF) and myeloid malignancies, characterized by somatic TP53-mutated clones and erythroid predominance. We present a series of 52 subjects (35 families) with ERCC6L2 biallelic germ line variants collected retrospectively from 11 centers globally, with a follow-up of 1165 person-years. At initial investigations, 32 individuals were diagnosed with BMF and 15 with a hematological malignancy (HM). The subjects presented with 19 different variants of ERCC6L2, and we identified a founder mutation, c.1424delT, in Finnish patients. The median age of the subjects at baseline was 18 years (range, 2-65 years). Changes in the complete blood count were mild despite severe bone marrow (BM) hypoplasia and somatic TP53 mutations, with no significant difference between subjects with or without HMs. Signs of progressive disease included increasing TP53 variant allele frequency, dysplasia in megakaryocytes and/or erythroid lineage, and erythroid predominance in the BM morphology. The median age at the onset of HM was 37.0 years (95% CI, 31.5-42.5; range, 12-65 years). The overall survival (OS) at 3 years was 95% (95% CI, 85-100) and 19% (95% CI, 0-39) for patients with BMF and HM, respectively. Patients with myelodysplastic syndrome or acute myeloid leukemia with mutated TP53 undergoing hematopoietic stem cell transplantation had a poor outcome with a 3-year OS of 28% (95% CI, 0-61). Our results demonstrated the importance of early recognition and active surveillance in patients with biallelic germ line ERCC6L2 variants.

Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia.

Relapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

A survey on thromboprophylaxis and coagulation assessment in children and young adults with acute lymphoblastic leukaemia (ALL) in the Nordic and Baltic countries: Different practices of assessment and management.

Patients undergoing treatment for acute lymphoblastic leukaemia (ALL) are at risk of coagulopathy, especially thromboembolism. We conducted a survey on practices in the assessment and management of coagulopathy during the new ALLTogether protocol in 29 (17 paediatric, 12 adult) Nordic and Baltic cancer centres. While 92% of adult centres used thromboprophylaxis with low-molecular-weight heparin, no paediatric centre did. Almost all providers performed baseline coagulation studies, but only 59% continued the assessment. Fibrinogen replacement was conducted in 59%, and antithrombin replacement in 28% of the centres. The survey highlights the need for guidelines in the management of coagulopathy during ALL therapy.

Incidence trends of childhood central nervous system tumors in Finland 1990-2017.

INTRODUCTION: Central nervous system (CNS) tumors are a leading cause of cancer-related morbidity and mortality in children. Our aim is to characterize incidence trends of pediatric CNS tumors in Finland over the last three decades. METHODS: Data on all benign and malignant incident CNS tumors diagnosed in children aged 0-14 years in 1990-2017 were extracted from the Finnish Cancer Registry and classified according to the 2016 WHO classification of CNS tumors. We analyzed age-standardized incidence rates (ASR) for pediatric CNS tumors overall and by sex, age, tumor histology, grade, and location using Poisson regression. We used joinpoint regression to evaluate changes in trends. RESULTS: Overall, 1117 pediatric CNS tumor cases were registered in Finland with a 1.2:1 male to female ratio. The average annual ASR was 4.3 per 100,000 person-years (95% CI 4.26, 4.34). The most common tumor type was pilocytic astrocytoma (30% of tumors), followed by medulloblastoma (10%) with incidence rates of 1.30 and 0.45 per 100,000 person-years, respectively. The overall incidence of pediatric CNS tumors increased by an annual percentage change (APC) of 0.8% (95% CI 0.2, 1.4). We observed no major changes in incidence trends of tumor histology groups or tumor location groups. The ASR of benign tumors increased by an APC of 1.0 (95% CI 0.1, 2.0). CONCLUSIONS: Utilizing the high-quality and completeness of data in the Finnish Cancer registry, we found that the incidence of pediatric CNS tumors in Finland has increased slightly from 1990 until 2017. Although variations in diagnostic and registration practices over time might have affected the rates, the trend may also reflect a true increase in incidence.

Trends in ferritin measurements in children and adolescents: A Finnish 9-year observational study.

AIM: A lack of stored iron, indicated by low serum ferritin, has been associated with various clinical symptoms. There are no longitudinal data on the frequency of ferritin measurements in children and adolescents. METHODS: A total of 2834 children aged <18 years with serum ferritin and other anaemia-related blood parameters taken during an outpatient visit between 2012 and 2019 were investigated. Patients with acute infections were excluded. Nationwide temporal and regional variations and correlations with public information searches through Google were analysed. RESULTS: A significant increase in the frequency of ferritin measurements was seen starting in 2018, with a 47-fold rise in 2019 compared to 2012. A simultaneous escalation in Google Search activity was seen. Deficiency of stored iron was relatively common: 21.6% of children with normal haemoglobin and 14.9% of non-anaemic children with normal red cell indices exhibited ferritin levels below 15 µg/L. CONCLUSION: Ferritin measurement has increased greatly among children and adolescents. Our results suggest that public interest and popular trends can significantly influence health care practices. This calls for further investigation into the causes and consequences of such a phenomenon. Prospective randomised intervention studies are needed to evaluate the utility of iron supplementation in patients with low iron storage levels.

Late adverse effects of childhood acute lymphoblastic leukemia treatment on developing dentition.

BACKGROUND: Childhood cancer survivors show a variety of late adverse effects on dental health. The purpose of this study was to examine the prevalence and severity of dental abnormalities in permanent dentition in childhood leukemia survivors. MATERIALS AND METHODS: Retrospective analysis of panoramic radiographs was performed for 178 childhood leukemia survivors aged below 17 years at the time of diagnosis. Sex, age at diagnosis, interval between ALL diagnosis and the follow-up radiograph, treatment protocol, and risk grouping were recorded. Abnormalities of tooth development and defect index were used to assess the frequency and severity of dental abnormalities. RESULTS: One hundred eight (61%) patients had no dental abnormalities at follow-up examination at a median of 6.1 years after diagnosis. Microdontia was more frequent in children under 6 years of age at the time of diagnosis (5.7% vs. 0.6%, p < .001). Significant differences were noted between distinct ALL treatment protocols with more common microdontia in patients treated according to the NOPHO ALL2008 protocol. Tooth agenesis was more frequent in patients that underwent therapy according to high-risk arms compared to intermediate- or standard-risk arms (3.8% vs. 1.4%, p = .01). Patients under 6 years of age at diagnosis had a significantly higher average defect index score than older patients (7.0 vs. 2.8, p = .01). CONCLUSIONS: Children and adolescents who received ALL treatment were at risk for dental damage. Young age and high-intensity therapy were associated with the severity of dental abnormalities.

Data-driven characterization of molecular phenotypes across heterogeneous sample collections.

Existing large gene expression data repositories hold enormous potential to elucidate disease mechanisms, characterize changes in cellular pathways, and to stratify patients based on molecular profiles. To achieve this goal, integrative resources and tools are needed that allow comparison of results across datasets and data types. We propose an intuitive approach for data-driven stratifications of molecular profiles and benchmark our methodology using the dimensionality reduction algorithm t-distributed stochastic neighbor embedding (t-SNE) with multi-study and multi-platform data on hematological malignancies. Our approach enables assessing the contribution of biological versus technical variation to sample clustering, direct incorporation of additional datasets to the same low dimensional representation, comparison of molecular disease subtypes identified from separate t-SNE representations, and characterization of the obtained clusters based on pathway databases and additional data. In this manner, we performed an integrative analysis across multi-omics acute myeloid leukemia studies. Our approach indicated new molecular subtypes with differential survival and drug responsiveness among samples lacking fusion genes, including a novel myelodysplastic syndrome-like cluster and a cluster characterized with CEBPA mutations and differential activity of the S-adenosylmethionine-dependent DNA methylation pathway. In summary, integration across multiple studies can help to identify novel molecular disease subtypes and generate insight into disease biology.

Randomized controlled and double-blinded study of Caphosol versus saline oral rinses in pediatric patients with cancer.

BACKGROUND: Oral mucositis (OM) is a significant side effect of cancer treatment. The purpose of this study was to compare topically administered Caphosol to saline rinses in the prevention of mucositis in pediatric cancer patients. PROCEDURE: A controlled, double-blinded, and randomized clinical crossover study recruited patients between 2 to 17.99 years of age who were diagnosed with a malignancy and were receiving either high-dose methotrexate (≥1 g/m2 ), anthracycline, or cisplatin chemotherapy (NCT0280733). All patients received two 7-day cycles of the mouth rinses; that is, one cycle of Caphosol and one cycle of saline in a randomized order. Oral changes and symptoms were evaluated using the World Health Organisation (WHO) toxicity scale and the Children's International Mucositis Evaluation Scale (ChIMES). The primary endpoint was the frequency and severity of OM and oral symptoms. RESULTS: A total of 56 patients were recruited to the study, of whom 45 were randomized with a median age of 6.5 years (range 2.1-17.1 years). No cases of severe OM were observed. Grade ≥ 3 oral symptoms were present at least once in six (13%) patients during the Caphosol cycle and 13 (29%) patients during the saline cycle (P = .12). The peak of symptom scores was evident at around day 4-7 after administration of the chemotherapy with no marked differences between the rinse solutions. Multivariable regression analysis did not indicate a benefit of using Caphosol over the saline solution. CONCLUSIONS: No difference in prevention of oral mucositis was observed between the use of Caphosol or saline rinses.

Nursing intensity scores did not correlate well with reimbursement claims for infant bronchiolitis.

AIM: We retrospectively evaluated the hospital reimbursement rates for inpatient bronchiolitis treatment and then compared them to the RAFAELA® nursing intensity scores. METHODS: We selected all 44 bronchiolitis patients treated in the paediatric intensive care unit (PICU) and then for each PICU-treated patient two patients treated on the ward (n = 88) under 12 months of age in 2010-2015. The data included medical histories, hospital reimbursement rates using the Nordic Diagnosis Related Groups (NordDRG) or expense categories and the RAFAELA® scores. RESULTS: Reimbursement claims were mostly based on expense categories for PICU admissions and NordDRG categories for ward admissions. The median (range) was €6352 (€1330-30 554) and €2009 (€768-6027) per episode for the PICU and ward cases, respectively. The median lengths of hospital stay were 8.5 days (3-18) and 3 days (1-8), respectively. Higher RAFAELA® scores were associated with nasal continuous positive airway pressure therapy and mechanical ventilation in the PICU and oxygen supplementation and nasogastric tube feeding on the ward. The correlation coefficients between RAFAELA® scores and hospital reimbursement claims ranged from 0.121-0.450. CONCLUSION: Hospital reimbursement claim for a PICU admission was three times as much as a ward admission and reimbursement claims for bronchiolitis did not match with nursing intensity scores.

Genome-wide repression of eRNA and target gene loci by the ETV6-RUNX1 fusion in acute leukemia.

Approximately 20%-25% of childhood acute lymphoblastic leukemias carry the ETV6-RUNX1 (E/R) fusion gene, a fusion of two central hematopoietic transcription factors, ETV6 (TEL) and RUNX1 (AML1). Despite its prevalence, the exact genomic targets of E/R have remained elusive. We evaluated gene loci and enhancers targeted by E/R genome-wide in precursor B acute leukemia cells using global run-on sequencing (GRO-seq). We show that expression of the E/R fusion leads to widespread repression of RUNX1 motif-containing enhancers at its target gene loci. Moreover, multiple super-enhancers from the CD19+/CD20+-lineage were repressed, implicating a role in impediment of lineage commitment. In effect, the expression of several genes involved in B cell signaling and adhesion was down-regulated, and the repression depended on the wild-type DNA-binding Runt domain of RUNX1. We also identified a number of E/R-regulated annotated and de novo noncoding genes. The results provide a comprehensive genome-wide mapping between E/R-regulated key regulatory elements and genes in precursor B cell leukemia that disrupt normal B lymphopoiesis.

Insulin-dependent diabetes: A chronic complication to acute pancreatitis in childhood acute lymphoblastic leukemia.

Pancreatitis is a frequent toxicity to acute lymphoblastic leukemia (ALL) treatment, significantly associated with asparaginase use, and may be followed by severe complications such as acute hyperglycaemia, need for mechanical ventilation, pseudocysts, and death. Here, we provide novel data on seven patients diagnosed with diabetes after pancreatitis and still requiring insulin treatment after a median follow-up of 4.2 years (range: 1.7-9.2). We describe the clinical course of pancreatitis and illustrate the association between pancreatic pseudocysts, older age, and development of insulin-dependent diabetes. Together, this study documents the persisting burden of pancreatitis in childhood ALL and underlines the need for plasma glucose level monitoring.

Parental occupational exposure to low-frequency magnetic fields and risk of leukaemia in the offspring: findings from the Childhood Leukaemia International Consortium (CLIC).

OBJECTIVES: Previously published studies on parental occupational exposure to extremely low-frequency magnetic fields (ELF-MF) and risk of acute lymphoblastic leukaemia (ALL) and acute myeloid leukaemia (AML) in their offspring were inconsistent. We therefore evaluated this question within the Childhood Leukemia International Consortium. METHODS: We pooled 11 case-control studies including 9723 childhood leukaemia cases and 17 099 controls. Parental occupational ELF-MF exposure was estimated by linking jobs to an ELF-MF job-exposure matrix (JEM). Logistic regression models were used to estimate ORs and 95% CIs in pooled analyses and meta-analyses. RESULTS: ORs from pooled analyses for paternal ELF-MF exposure >0.2 microtesla (µT) at conception were 1.04 (95% CI 0.95 to 1.13) for ALL and 1.06 (95% CI 0.87 to 1.29) for AML, compared with ≤0.2 µT. Corresponding ORs for maternal ELF-MF exposure during pregnancy were 1.00 (95% CI 0.89 to 1.12) for ALL and 0.85 (95% CI 0.61 to 1.16) for AML. No trends of increasing ORs with increasing exposure level were evident. Furthermore, no associations were observed in the meta-analyses. CONCLUSIONS: In this large international dataset applying a comprehensive quantitative JEM, we did not find any associations between parental occupational ELF-MF exposure and childhood leukaemia.

Acute Lymphoblastic Leukemia With INPP5D-ABL1 Fusion Responds to Imatinib Treatment.

We describe a patient with Down syndrome whose precursor B-cell acute lymphoblastic leukemia cells expressed INPP5D-ABL1 fusion gene that resulted in a reciprocal chromosome translocation t(2;9)(q27;q34). The fusion gene was present as a small subclone in the primary disease but was first identified at relapse when the subclone had expanded into a major clone. At relapse, the patient responded poorly to conventional induction chemotherapy but a transient morphologic remission was achieved after administration of imatinib monotherapy. This case demonstrates a pathway to relapse in a Down syndrome patients with acute lymphoblastic leukemia through a rare fusion event. It highlights the significance of minor subclonal events in therapy resistance and the opportunity provided for targeted therapy.

Radiation exposure from computerized tomography and risk of childhood leukemia: Finnish register-based case-control study of childhood leukemia (FRECCLE).

The only well-established risk factors for childhood leukemia are high-dose ionizing radiation and Down syndrome. Computerized tomography is a common source of low-dose radiation. In this study, we examined the magnitude of the risk of childhood leukemia after pediatric computed tomography examinations. We evaluated the association of computed tomography scans with risk of childhood leukemia in a nationwide register-based case-control study. Cases (n=1,093) were identified from the population-based Finnish Cancer Registry and three controls, matched by gender and age, were randomly selected for each case from the Population Registry. Information was also obtained on birth weight, maternal smoking, parental socioeconomic status and background gamma radiation. Data on computed tomography scans were collected from the ten largest hospitals in Finland, covering approximately 87% of all pediatric computed tomography scans. Red bone marrow doses were estimated with NCICT dose calculation software. The data were analyzed using exact conditional logistic regression analysis. A total of 15 cases (1.4%) and ten controls (0.3%) had undergone one or more computed tomography scans, excluding a 2-year latency period. For one or more computed tomography scans, we observed an odds ratio of 2.82 (95% confidence interval: 1.05 - 7.56). Cumulative red bone marrow dose from computed tomography scans showed an excess odds ratio of 0.13 (95% confidence interval: 0.02 - 0.26) per mGy. Our results are consistent with the notion that even low doses of ionizing radiation observably increase the risk of childhood leukemia. However, the observed risk estimates are somewhat higher than those in earlier studies, probably due to random error, although unknown predisposing factors cannot be ruled out.

Effects of incomplete residential histories on studies of environmental exposure with application to childhood leukaemia and background radiation.

When evaluating environmental exposures, residential exposures are often most relevant. In most countries, it is impossible to establish full residential histories. In recent publications, childhood leukaemia and background radiation have been studied with and without full residential histories. This paper investigates the consequences of lacking such full data. Data from a nationwide Finnish Case-Control study of Childhood Leukaemia and gamma rays were analysed. This included 1093 children diagnosed with leukaemia in Finland in 1990-2011. Each case was matched by gender and year of birth to three controls. Full residential histories were available. The dose estimates were based on outdoor background radiation measurements. The indoor dose rates were obtained with a dwelling type specific conversion coefficient and the individual time-weighted mean red bone marrow dose rates were calculated using age-specific indoor occupancy and the age and gender of the child. Radiation from Chernobyl fallout was included and a 2-year latency period assumed. The median separation between successive dwellings was 3.4 km and median difference in red bone marrow dose 2.9 nSv/h. The Pearson correlation between the indoor red bone marrow dose rates of successive dwellings was 0.62 (95% CI 0.60, 0.64). The odds ratio for a 10 nSv/h increase in dose rate with full residential histories was 1.01 (95% CI 0.97, 1.05). Similar odds ratios were calculated with dose rates based on only the first dwelling (1.02, 95% CI 0.99, 1.05) and only the last dwelling (1.00, 95% CI 0.98, 1.03) and for subjects who had lived only in a single dwelling (1.05, 95% CI 0.98, 1.10). Knowledge of full residential histories would always be the option of choice. However, due to the strong correlation between exposure estimates in successive dwellings and the uncertainty about the most relevant exposure period, estimation of overall exposure level from a single address is also informative. Error in dose estimation is likely to cause some degree of classical measurement error resulting in bias towards the null.

Deregulation of the non-coding genome in leukemia.

Methodological advances that allow deeper characterization of non-coding elements in the genome have started to reveal the full spectrum of deregulation in cancer. We generated an inducible cell model to track transcriptional changes after induction of a well-known leukemia-inducing fusion gene, ETV6-RUNX1. Our data revealed widespread transcriptional alterations outside coding elements in the genome. This adds to the growing list of various alterations in the non-coding genome in cancer and pinpoints their role in diseased cellular state.