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Background

Introduction

Introduction:

Mutation status of FLT3, NPM1, CEBPA, and WT1genes and gene expression levels of ERG, MN1, BAALC, FLT3, and WT1have been identified as possible prognostic markers in acute myeloid leukemia (AML). We have performed a thorough prognostic evaluation of these genetic markers in patients with pediatric AML enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) 1993 or NOPHO 2004 protocols. Mutation status and expression levels were analyzed in 185 and 149 patients, respectively. Presence of FLT3-internal tandem duplication (ITD) was associated with significantly inferior event-free survival (EFS), whereas presence of an NPM1mutation in the absence of FLT3-ITD correlated with significantly improved EFS. Furthermore, high levels of ERGand BAALCtranscripts were associated with inferior EFS. No significant correlation with survival was seen for mutations in CEBPAand WT1or with gene expression levels of MN1, FLT3, and WT1. In multivariate analysis, the presence of FLT3-ITD and high BAALCexpression were identified as independent prognostic markers of inferior EFS. We conclude that analysis of the mutational status of FLT3and NPM1at diagnosis is important for prognostic stratification of patients with pediatric AML and that determination of the BAALCgene expression level can add valuable information.

Mutation status of FLT3, NPM1, CEBPA, and WT1 genes and gene expression levels of ERG, MN1, BAALC, FLT3, and WT1 have been identified as possible prognostic markers in acute myeloid leukemia (AML). We have performed a thorough prognostic evaluation of these genetic markers in patients with pediatric AML enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) 1993 or NOPHO 2004 protocols. Mutation status and expression levels were analyzed in 185 and 149 patients, respectively. Presence of FLT3-internal tandem duplication (ITD) was associated with significantly inferior event-free survival (EFS), whereas presence of an NPM1 mutation in the absence of FLT3-ITD correlated with significantly improved EFS. Furthermore, high levels of ERG and BAALC transcripts were associated with inferior EFS. No significant correlation with survival was seen for mutations in CEBPA and WT1 or with gene expression levels of MN1, FLT3, and WT1. In multivariate analysis, the presence of FLT3-ITD and high BAALC expression were identified as independent prognostic markers of inferior EFS. We conclude that analysis of the mutational status of FLT3 and NPM1 at diagnosis is important for prognostic stratification of patients with pediatric AML and that determination of the BAALC gene expression level can add valuable information.

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.

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.

The t(12;21) (p13;q22) translocation resulting in ETV6/RUNX1 (previously named TEL/AML1) gene fusion is present in about 25% of children with precursor B-lineage acute lymphoblastic leukemia (B-ALL). We successfully tested 275 precursor B-ALL samples from children aged 1 to 17 years to determine the relation between t(12;21) and in vitro cellular drug resistance, measured by the fluorometric microculture cytotoxicity assay (FMCA). Samples from 83 patients (30%) were positive for t(12;21). The ETV6/RUNX1+samples were significantly more sensitive than ETV6/RUNX1–samples to doxorubicin, etoposide, amsacrine, and dexamethasone, whereas the opposite was true for cytarabine. After matching for unevenly distributed patient characteristics, that is, excluding patients with high hyperdiploidy (> 51 chromosomes), t(9; 22), t(1;19), or 11q23 rearrangement, the ETV6/RUNX1+samples remained significantly more sensitive to doxorubicin (P= .001) and etoposide (P= .001). For the other drugs tested (amsacrine, cytarabine, dexamethasone, prednisolone, vincristine, 6-thioguanine, and 4-hydroperoxy-cyclophosphamide), no significant difference in cellular drug sensitivity was found. In conclusion, we found that the presence of the t(12;21) translocation in childhood precursor B-ALL is associated with a high tumor cell sensitivity to doxorubicin and etoposide. High throughput techniques should now be used to elucidate the cellular mechanisms by which ETV6/RUNX1 gene fusion is linked to increased sensitivity to these drugs.

The t(12;21) (p13;q22) translocation resulting in ETV6/RUNX1 (previously named TEL/AML1) gene fusion is present in about 25% of children with precursor B-lineage acute lymphoblastic leukemia (B-ALL). We successfully tested 275 precursor B-ALL samples from children aged 1 to 17 years to determine the relation between t(12;21) and in vitro cellular drug resistance, measured by the fluorometric microculture cytotoxicity assay (FMCA). Samples from 83 patients (30%) were positive for t(12;21). The ETV6/RUNX1+ samples were significantly more sensitive than ETV6/RUNX1– samples to doxorubicin, etoposide, amsacrine, and dexamethasone, whereas the opposite was true for cytarabine. After matching for unevenly distributed patient characteristics, that is, excluding patients with high hyperdiploidy (> 51 chromosomes), t(9; 22), t(1;19), or 11q23 rearrangement, the ETV6/RUNX1+ samples remained significantly more sensitive to doxorubicin (P = .001) and etoposide (P = .001). For the other drugs tested (amsacrine, cytarabine, dexamethasone, prednisolone, vincristine, 6-thioguanine, and 4-hydroperoxy-cyclophosphamide), no significant difference in cellular drug sensitivity was found. In conclusion, we found that the presence of the t(12;21) translocation in childhood precursor B-ALL is associated with a high tumor cell sensitivity to doxorubicin and etoposide. High throughput techniques should now be used to elucidate the cellular mechanisms by which ETV6/RUNX1 gene fusion is linked to increased sensitivity to these drugs.

• Twenty 2- to 13-year-old infantile autistic children (16 boys and four girls) and four 4- to 13-year-old children (two boys and two girls) with other kinds of childhood psychoses were compared with eight 6-month-old to 6-year-old normal children with regard to cerebrospinal fluid contents of endorphin fractions I and II. The psychosis groups showed higher mean cerebrospinal fluid endorphin fraction II levels, and 11 (55%) of the 20 autistic patients showed values higher than the highest in the group of normal children. There was a trend toward a correlation between high fraction II levels and self-destructiveness and decreased pain sensibility in the psychotic children. The results are regarded as preliminary but as warranting further research in this potentially fruitful field.

Summary: Lung tissue from human fetuses, with gestational ages ranging between 14–26 wk, was studied by histochemical and biochemical methods. The findings were similar in all tissues tested, without apparent correlation to gestational age. Staining that indicated carbonic anhydrase activity was found in the capillary endothelium and in the epithelium of some segments of the peripheral airways. The ciliated epithelium of the central airways was unstained. The distribution of the enzyme in the human fetal lung differed clearly from that in the adult human lung, where little or no enzyme has been found in the airway epithelium. The mean carbonic anhydrase activity in whole homogenates of fetal lung tissue was 24 enzyme units per g wet weight of tissue. Ninety % of this activity was recovered in the supernatant fraction. Assay of this fraction by a radioimmunosorbent technique showed the presence of the carbonic anhydrase isoenzyme HCA-C corresponding to 380 ng enzyme per mg tissue protein. Small amounts of HCA-B were also found but are thought to be attributable to contaminating erythrocytes; thus, the data suggest that both the capillary endothelium and the lung epithelium contain HCA-C, an isoenzyme of carbonic anhydrase known to be involved in electrolyte transport in many tissues.Speculation: Carbonic anhydrase is probably involved in lung liquid secretion in the fetal lamb. The present data suggest that this enzyme plays a similar role in the human fetal lung.

Summary: Kidney tissue from human fetuses of gestational ages ranging 12–26 wk were studied by histochemical and biochemical methods. Clear staining for carbonic anhydrase activity was found in both proximal and distal parts of some nephrons already at gestational age of 12–15 wk. The staining pattern in the inner cortex of the fetuses of 24 and 26 wk did not differ markedly from that in adult renal cortex, except for the absence of capillary staining; proximal and distal convoluted tubules as well as initial collecting tubules exhibited distinct enzyme activity. In the outer cortex, where the nephronogenic zone is located, newly formed nephrons with no or little staining were found in all kidneys. Loops of Henle were absent or sparse in the medulla of the fetuses of 12–17 wk. Fully developed loops of Henle were relatively few also in the fetuses of 24 and 26 wk; the thick ascending limb and part of the thin limb were stained in such loops. The collecting ducts in all kidneys contained a varying number of intensely stained cells interspersed among unstained or weakly stained ones.The catalytic activity and the immunoassayable amount of carbonic anhydrase were determined in homogenates of renal cortex from three fetuses (19,21, and 26 wk). The kidney from the fetus of 26 wk showed the highest values with an enzyme activity and an immunoassayable amount of HCA-C corresponding to 204 enzyme units and 0.14 mg enzyme per g wet weight of tissue, respectively. These values are similar to those of adult renal cortex and indicate that all of antigenically determined HCA-C is also catalytically active in the kidney at this gestational age.As judged from their content of carbonic anhydrase many nephrons might be fully able to reabsorb bicarbonate and secrete hydrogen ions in human fetuses of 24–26 wk. Other nephrons are much less developed at this stage, however. This nephronic heterogeneity might at least partly explain the reduced capacity for urinary acidification which has been reported for premature newborn infants.

The distribution of carbonic anhydrase in the metanephrogenic zone of the human fetal kidney was studied by the histochemical method of Hansson. In stage I nephrons (= renal vesicles) no or very little enzyme activity was found. Adjacent cortical collecting tubules, however, showed enzyme activity at the luminal cell surface. In stage II nephrons (= S-shaped tubules) the tubular cells displayed cytoplasmic carbonic anhydrase activity, while the glomeruli were inactive. Thus, the development of the nephron from stage I to stage II was accompanied by an increase in tubular cell carbonic anhydrase activity with partially another distribution than in the adult kidney. This may indicate an additional function of this enzyme in the developing nephron other than in the process of urinary acidification, e.g. to promote normal growth and differentiation of the tubular cells.

Kidney tissue from human fetuses of gestational ages ranging 12–26 wk were studied by histochemical and biochemical methods. Clear staining for carbonic anhydrase activity was found in both proximal and distal parts of some nephrons already at gestational age of 12–15 wk. The staining pattern in the inner cortex of the fetuses of 24 and 26 wk did not differ markedly from that in adult renal cortex, except for the absence of capillary staining; proximal and distal convoluted tubules as well as initial collecting tubules exhibited distinct enzyme activity. In the outer cortex, where the nephronogenic zone is located, newly formed nephrons with no or little staining were found in all kidneys. Loops of Henle were absent or sparse in the medulla of the fetuses of 12–17 wk. Fully developed loops of Henle were relatively few also in the fetuses of 24 and 26 wk; the thick ascending limb and part of the thin limb were stained in such loops. The collecting ducts in all kidneys contained a varying number of intensely stained cells interspersed among unstained or weakly stained ones.

Lung tissue from human fetuses, with gestational ages ranging between 14–26 wk, was studied by histochemical and biochemical methods. The findings were similar in all tissues tested, without apparent correlation to gestational age. Staining that indicated carbonic anhydrase activity was found in the capillary endothelium and in the epithelium of some segments of the peripheral airways. The ciliated epithelium of the central airways was unstained. The distribution of the enzyme in the human fetal lung differed clearly from that in the adult human lung, where little or no enzyme has been found in the airway epithelium. The mean carbonic anhydrase activity in whole homogenates of fetal lung tissue was 24 enzyme units per g wet weight of tissue. Ninety of this activity was recovered in the supernatant fraction. Assay of this fraction by a radioimmunosorbent technique showed the presence of the carbonic anhydrase isoenzyme HCA-C corresponding to 380 ng enzyme per mg tissue protein. Small amounts of HCA-B were also found but are thought to be attributable to contaminating erythrocytes; thus, the data suggest that both the capillary endothelium and the lung epithelium contain HCA-C, an isoenzyme of carbonic anhydrase known to be involved in electrolyte transport in many tissues.

AbstractAmpicillin was administered intramuscularly and amoxycillin or pivampicillin orally to 14 fasting newborn infants, 6–13 days old, in a cross-over trial. The dose was 50 mgkg twice daily. The mean peak plasma level of amoxycillin and pivampicillin was 58 (range 35–96) and 48 (33–82) of that noted after i.m. ampicillin, which gave a value of about 44±5 μgml (mean ± SEM) in both groups. The area under the time-concentration curve was 75 (range 60–101) of that of i.m. ampicillin for amoxycillin and 51 (20–76) for pivampicillin (p<0.05). Both drugs, especially amoxycillin, should be useful for oral treatment of neonatal infections caused by susceptible microorganisms in infants who are not critically ill.

Tissues from human fetuses with a gestational age of 19–26 weeks were studied by histochemical and biochemical methods. In gastric homogenates both the catalytic activity and the immunoassayable amount of carbonic anhydrase increased with gestational age. The enzyme activity and isoenzyme pattern in a fetus of 26 weeks were similar to those found in adult gastric mucosa. High enzyme activity was demonstrated histochemically in the gastric surface epithelium at a gestational age of 19 weeks. At this age, the number of stained parietal cells was low, but it increased considerably with gestational age. In all fetal gastrointestinal tissues tested there was a total lack of capillary staining for carbonic anhydrase activity, which contrasts to the clear staining found in adult tissues. The amounts of carbonic anhydrase in the small intestine were lower in the fetuses than in adults, especially in the jejunum; the difference was greater for the isoenzyme HCA-B than for HCA-C. Histochemically, the staining of jejunal epithelial cells in the fetuses was clearly reduced, both on the villi and in the crypts. In the ileum, single epithelial cells on the villi were distinctly stained, a finding similar to that in adult ileal epithelium. The colon showed the most striking differences between fetal and adult tissues, with much lower levels of both isoenzymes HCA-B and HCA-C and less staining of the epithelium in the fetal colon. Thus, the developmental pattern of carbonic anhydrase varied considerably among the intestinal tissues. The functional significance of the differences between fetal and adult tissues is only partly understood at present.

Plasma concentrations of 6-mercaptopurine (6-MP) were determined by gas chromatography-mass spectrometry. Ten children (nine with acute lymphatic leukemia) were studied on 2 consecutive days after oral intake of 6-MP. On one day the drug was administered in the fasting state and on the other (in random order) together with breakfast. The peak plasma concentrations of 6-MP after the dose intake with breakfast in percent of that in the fasting state (meal in % of fasting for each individual) varied between 33 and 181% (mean 111), and the area under the plasma concentration-time curve varied between 47 and 186% (mean 103). Thus, there were considerable variations among patients, but, for the group as a whole, there were no statistically significant differences between the two experimental conditions. This study cannot therefore form the basis for a recommendation as to whether 6-MP should be administered on an empty stomach or together with food.

Plasma and erythrocyte concentrations of 6-mercaptopurine (6-MP) were determined by gas chromatography-mass spectrometry. Eleven children (9 with acute lymphatic leukemia) were studied after oral intake of 6-MP doses ranging between 31 and 128 mg/m2 body surface area. The concentrations of 6-MP in plasma were found to vary considerably between patients even after dose normalization to 75 mg/m2. After dose normalization the mean peak plasma concentration was 0.68 μM (range 0.12-1.38) and the area under the plasma concentration-time curve (AUC) was 1.37 μM.h (range 0.12-3.04). The mean time taken to reach the peak concentration was 1.3 h (range 1-2), and the half-life of elimination was 1.8 h (range 0.6-2.5). No patient had detectable 6-MP concentrations 12 h after dose intake. The concentrations of 6-MP tended to be higher in erythrocytes than in plasma. The mean peak concentration in erythrocytes was 131% and the AUC 145% of that found in plasma. The mean half-life of elimination from erythrocytes was 2.0 h (range 0.7-2.8). These data indicate that 6-MP can pass through cell membranes rapidly to reach intracellular concentrations equal to or even higher than in plasma. In summary, marked interindividual differences in pharmacokinetics were found, probably due to highly variable bioavailability of oral 6-MP. Further studies are needed to determine whether measurements of plasma concentrations of 6-MP can be used to optimize maintenance treatment of childhood leukemia.

No relevant conflicts of interest to declare.

Background:Next generation sequencing allows for the detection of expressed fusion transcripts across the transcriptome and has spurred the discovery of many novel chimeric transcripts in various cancers. Structural chromosomal rearrangements that lead to fusion transcripts are a hallmark of acute lymphoblastic leukemia (ALL) and serve as markers for diagnosis and stratification of pediatric ALL patients into prognostically relevant subgroups. Improved delineation of structural alterations in ALL could provide additional information for prognosis in ALL and for improved stratification of patients into treatment groups.

We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. We used the methylation status of ~450,000 CpG sites from 546 well-characterized patients with T-ALL or B-cell precursor ALL with the cytogenetic subtypes high hyperdiploidy (HeH), t(12;21)(p13;q22)[ETV6/RUNX1], t(1;19)(q23;p13.3)[TCF3/PBX1], 11q23/MLL-rearrangements, t(9;22)(q34;q11)[BCR/ABL1], dic(9;20)(p13.2;q11.2), and intrachromosomal amplification of chromosome 21 iAMP21[RUNX1x >3], to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived consisting of only 246 CpG sites that accurately predicted the subtype of >96% of the patients with known subtype. The mean sensitivity and specificity across the subtypes was 0.90 and 0.99, respectively. We then used the DNA methylation classifier to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations (“other” subtype). Nearly half (n=106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative PCR and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1,and PAX5. The CpG sites constituting the classifiers highlight relevant biological characteristics of otherwise unclassified ALL patients and provide clues to the origin and development of leukemic transformation. Together with response to induction therapy, genetic aberrations are among the most important prognostic factors in pediatric ALL. Our findings indicate that DNA methylation profiling can help clarify some of the heterogeneity in cytogenetically undefined ALL patient groups and can potentially be implemented for diagnostics of ALL and other types of hematological cancers.

No relevant conflicts of interest to declare.

Although aberrant DNA methylation has been observed previously in acute lymphoblastic leukemia (ALL), the patterns of differential methylation have not been comprehensively determined in all subtypes of ALL on a genome-wide scale. The relationship between DNA methylation, cytogenetic background, drug resistance and relapse in ALL is poorly understood.