Pediatric acute myeloid leukemia (AML) is the second most common type of pediatric leukemia. Patients with AML are at high risk for several complications such as infections, typhlitis, and acute and ...long-term cardiotoxicity. Despite this knowledge, there are no definite supportive care guidelines as to what the best approach is to manage or prevent these complications.
The NOPHO-DB-SHIP (Nordic-Dutch-Belgian-Spain-Hong-Kong-Israel-Portugal) consortium, in preparation for a new trial in pediatric AML patients, had dedicated meetings for supportive care. In this review, the authors discuss the available data and outline recommendations for the management of children and adolescents with AML with an emphasis on hyperleukocytosis, tumor lysis syndrome, coagulation abnormalities and bleeding, infection, typhlitis, malnutrition, cardiotoxicity, and fertility preservation.
Improved supportive care has significantly contributed to increased cure rates. Recommendations on supportive care are an essential part of treatment for this highly susceptible population and will further improve their outcome.
The prognosis of children with acute myeloid leukemia (AML) is improving but still unsatisfactory. We and others have shown that measurable residual disease (MRD) as measured with multiparameter flow ...cytometry (MFC) is a very strong prognostic factor, however this technique cannot reliably identify low-risk cases. An alternative approach for MRD analysis is quantification of leukemia-specific transcripts using reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR). This approach has shown strong prognostic value in adult AML but its significance in childhood AML is less studied. In this retrospective study, we evaluated early treatment response with RT-qPCR and MFC in parallel to determine treatment kinetics and the relationship between results of the two methods. The study included 15 children (7 females, 8 males, median age 6 years (range 1-16)), diagnosed 2004-2011 with de novo AML with a quantifiable fusion transcript (8 with RUNX1-RUNX1T1, one with CBFB-MYH11 and 6 with KMT2A-MLLT3), all treated in the NOPHO AML-2004 trial. MRD analyses were performed on samples from day 15 from start of the first induction course and just before the first consolidation course. MRD analysis with MFC was performed in bone marrow samples by identifying leukemia-associated immunophenotypes (LAIP), and reported as percent of viable cells. Fusion transcripts were analyzed with RT-qPCR in bone marrow and blood according to the Europe against cancer program, and reported as percentage of diagnostic level in bone marrow. Results of all MRD analyses were reported to clinicians but not used for treatment decisions. All patients achieved complete remission. 9 patients relapsed (median time from diagnosis 12 months, range 8-16); 5 with RUNX1-RUNX1T1 and 4 with KMT2A-MLLT3. When we compared results of RT-qPCR in blood and bone marrow day 15 and before consolidation, there was a high correlation; 0.93, p<0.01. When 0.1% was used as cut-off for positivity, 20/22 samples were concordant (Cohens kappa test K value 0.818, P<0.001) with the two disconcordant samples positive in bone marrow but negative in blood before consolidation. Of the concordant samples, 9 were ≥0.1% and 11 were <0.1% in both bone marrow and blood. There was no significant correlation between results obtained with MFC and RT-qPCR, neither in bone marrow nor in blood. When comparing MFC and RT-qPCR results in bone marrow using the 0.1% cut-off, only 12 of 22 samples were concordant (4/10 at day 15, 8/12 before consolidation, in total 55% agreement; K value 0.141, P=0.36). Most discordant results (9/10) were ≥0.1% with RT-qPCR but negative (<0.1%) with MFC. There was a pattern of slower treatment kinetics with higher MRD results obtained with RT-qPCR than MFC. In cases with RUNX1-RUNX1T1 fusion transcript levels were 22% (median, range 12-63%) of diagnostic value day 15 and 0.03% (0-1.4%) before consolidation. In cases with KMT2A/MLLT3, the corresponding levels were 15% (0 - 40%) and 0 (0-0.038%). There was no difference in relapse frequency between children with fusion transcripts ≥0.1% or <0.1% day 15. On the other hand, two patients with RUNX1-RUNX1T1 fusion transcript level ≥0.1% but negative with MFC before consolidation both relapsed. In order to find the reason for remaining high levels of fusion transcripts day 15 when the bone marrow is usually hypoplastic, we reanalyzed flow cytometry files from four cases with remaining fusion transcript >0.1%. These analyses confirmed the absence of immature cells with LAIP and detected either more mature myeloid cells or immature cells without LAIP. We hypothesize that fusion transcripts can be present in mature cells not detected as MRD using MFC. This was supported by analyzing sorted immature and mature cells from bone marrow samples at diagnosis of AML (n=4, adults). In CD34-CD117+ cells, leukemic transcripts (RUNX1-RUNX1T1/CBFB-MYH11/mutated NPM1) were 67±10% (mean±SEM) of the level in the most immature cells, CD34+CD117+, from the same patients and in mature granulocytes (CD34-CD117-, high SSC) 88±9%. In conclusion, RT-qPCR and MFC provide different information during induction treatment. RT-qPCR has a stronger ability to detect remaining leukemic burden, but whether this burden is clinically relevant remains to be shown. The impact of fusion transcript levels on relapse risk is currently investigated in the NOPHO-DBH AML-2012 trial in which MFC is used for risk stratification.
No relevant conflicts of interest to declare.
Relapse remains a major therapeutic challenge in children with acute myeloid leukemia (AML). Outcome after relapse may improve if preemptive therapy is initiated at first evidence of leukemia ...regrowth. Early detection of imminent relapse requires molecular measurable residual disease (MRD) monitoring after therapy completion. Today, this is possible only in about 40% of children with AML that harbor genetic abnormalities applicable for quantification using standardized qPCR assays. To enable disease surveillance for all patients, we developed patient-tailored deep sequencing (DS) MRD analysis, which provides highly sensitive detection of leukemia-specific mutations. We investigated the potential of this method for early relapse detection in peripheral blood (PB), the only easily accessible source for MRD sampling in children.
PB samples were collected at monthly intervals during follow-up from 45 children diagnosed with AML and treated according to The Nordic Society of Pediatric Haematology and Oncology (NOPHO)-DBH AML 2012 protocol between January 2013 and May 2016 in Denmark, Norway, Sweden and Finland (508 samples, median 11 samples/patient, range 3-27). Nine patients with relapse (median age 5 years, range 0-8) had available diagnostic and relapse material and were included in this study. The patients displayed core binding factor abnormalities (n=3), KMT2A-rearrangements (n=3), monosomy 7 (n=1) or normal karyotype (n=2) at AML diagnosis. Leukemia-specific single nucleotide variants (SNVs) were identified with exome sequencing (ES) of sorted leukemic cells with lymphocytes or remission PB as constitutive DNA template. A variant allele frequency (VAF) with 95% confidence interval including 50% indicates presence of the mutation in all leukemic cells at diagnosis. With the exception of 2 cases with only subclonal mutations at diagnosis, leukemia-specific SNVs with VAF of 50% at diagnosis and persistence at relapse were selected as MRD targets. MRD target mutations were quantified in PB samples preceding overt relapse using patient-tailored DS assays with sensitivity of VAF 0.02%. In diagnostic samples, ES identified 53 leukemia-specific SNVs (median 4 SNVs/patient, range 2-12) of which 33 were also present at relapse (median 2 SNVs/patient, range 1-9). The number of mutations identified at diagnosis increased with age (Rs 0.83, p=0.006). All patients had at least one leukemia-specific SNV detected at both diagnosis and relapse. Twenty-one MRD target mutations (median 2 SNVs/patient, range 1-3) were quantified in PB (55 samples, median sampling interval 28 days, range 11-80) using DS. In 8/9 patients, at least one SNV was detected in PB before overt relapse occurred. The first PB sample showing MRD positivity (median VAF 0.14%, range 0.03-0.44) preceded hematological relapse at a median interval of 3 months (range 0-7.9). In 6 patients not preemptively treated, the median doubling time based on VAF increments was 7 days, with great variability between individuals and genotypes (range 4-28 days). Three patients had molecular relapse diagnosed by qPCR used in clinical diagnostics and received individualized preemptive treatment. In these 3 patients, DS detected mutations in PB for >100 days preceding overt relapse and the doubling times were 14, 25 and 36 days.
In conclusion, DS of leukemia-specific mutations at frequent intervals in PB enables early detection of relapse and ES at diagnosis may identify SNVs applicable for such longitudinal MRD monitoring. This approach facilitates molecular disease surveillance and initiation of preemptive therapy in AML patients without established qPCR targets.
No relevant conflicts of interest to declare.
Background
Supportive‐care use of granulocyte colony‐stimulating factor (G‐CSF) in pediatric acute myeloid leukemia (AML) remains controversial due to a theoretical increased risk of relapse and ...limited impact on neutropenic complications. We describe the use of G‐CSF in patients treated according to NOPHO‐AML 2004 and DB AML‐01 and investigated associations with relapse.
Procedure
Patients diagnosed with de novo AML completing the first week of therapy and not treated with hematopoietic stem cell transplantation in the first complete remission were included (n = 367). Information on G‐CSF treatment after each course (yes/no) was registered prospectively in the study database and detailed information was gathered retrospectively from each center. Descriptive statistics were used to describe G‐CSF use and Cox regression to assess the association between G‐CSF and risk of relapse.
Results
G‐CSF as supportive care was given to 128 (35%) patients after 268 (39%) courses, with a large variation between centers (0‐93%). The use decreased with time—the country‐adjusted odds ratio was 0.8/diagnostic year (95% confidence interval CI 0.7‐0.9). The median daily dose was 5 μg/kg (range 3‐12 μg/kg) and the median cumulative dose was 75 μg/kg (range 7‐1460 μg/kg). Filgrastim was used in 82% of G‐CSF administrations and infection was the indication in 44% of G‐CSF administrations. G‐CSF was associated with increased risk of relapse—the adjusted hazard ratio was 1.5 (95% CI 1.1‐2.2).
Conclusions
G‐CSF as supportive care was used in a third of patients, and use decreased with time. Our results indicate that the use of G‐CSF may be associated with an increased risk of relapse.
Hyperleucocytosis in paediatric acute myeloid leukaemia (AML) is associated with increased morbidity and mortality. We studied hyperleucocytosis in 890 patients with AML aged 0-18 years registered in ...the Nordic Society of Paediatric Haematology and Oncology (NOPHO) registry, with special focus on very high white blood cell counts (WBC >200 × 10/l). Eighty-six patients (10%) had WBC 100-199 × 10
/l and 57 (6%) had WBC ≥200 × 10
/l. Patients with WBC ≥200 × 10
/l had a high frequency of t(9;11) and a paucity of trisomy 8. Due to the high frequency of deaths within the first 2 weeks (30% vs. 1% for all others), overall survival in this group was inferior to patients with WBC <200 × 10
/l (39% vs. 61%). Main cause of early death was intracranial haemorrhage and leucostasis. Twenty-six per cent of these patients never started antileukaemic protocol therapy. Leukapheresis or exchange transfusion was used in 24% of patients with hyperleucocytosis without impact on survival. Patients with hyperleucocytosis surviving the first week had identical survival as patients with lower WBC. We conclude that death within the first days after diagnosis is the major challenge in patients with high WBC and advocate rapid initiation of intensive chemotherapy.
Summary Hyperleucocytosis in paediatric acute myeloid leukaemia (AML) is associated with increased morbidity and mortality. We studied hyperleucocytosis in 890 patients with AML aged 0-18 years ...registered in the Nordic Society of Paediatric Haematology and Oncology (NOPHO) registry, with special focus on very high white blood cell counts (WBC >200 × 10/l). Eighty-six patients (10%) had WBC 100-199 × 109/l and 57 (6%) had WBC ≥200 × 109/l. Patients with WBC ≥200 × 109/l had a high frequency of t(9;11) and a paucity of trisomy 8. Due to the high frequency of deaths within the first 2 weeks (30% vs. 1% for all others), overall survival in this group was inferior to patients with WBC <200 × 109/l (39% vs. 61%). Main cause of early death was intracranial haemorrhage and leucostasis. Twenty-six per cent of these patients never started antileukaemic protocol therapy. Leukapheresis or exchange transfusion was used in 24% of patients with hyperleucocytosis without impact on survival. Patients with hyperleucocytosis surviving the first week had identical survival as patients with lower WBC. We conclude that death within the first days after diagnosis is the major challenge in patients with high WBC and advocate rapid initiation of intensive chemotherapy.
Background: Long-term patient survival in childhood acute myeloid leukemia (AML) has improved dramatically the last decades. However, 30% of children with AML die from the disease with hematological ...relapse being the most common event and cause of treatment failure. Early detection of imminent hematological relapse during follow-up may facilitate preparation of upcoming therapy including hematopoietic stem cell transplantation (HSCT) or an intervention e.g. preemptive therapy. Increasing levels of minimal residual disease (MRD) in peripheral blood (PB) may predict impending relapse in patients in first complete remission (CR1). Quantitative PCR (qPCR) is a highly sensitive assay suitable for MRD quantification in PB but absence and instability of valid qPCR MRD markers limit molecular detection of disease recurrence in a large proportion of patients. The Wilms tumor 1 gene (WT1) is frequently overexpressed at diagnosis in hematological malignancies but the predictive value of WT1 expression levels in childhood AML during follow-up remain uncertain.
We investigated the applicability of various fusion transcripts and WT1 expression as putative MRD targets in PB disease monitoring.
Methods: From January 2004 to August 2014, 135 pediatric patients were diagnosed with de novo AML and treated on two consecutive protocols, NOPHO-AML 2004 and NOPHO-DBH AML 2012, in Iceland, Finland and Denmark. Potential qPCR MRD targets were identified in 69% (93/135) of these patients. qPCR MRD were quantified in PB at scheduled time points during therapy followed by sampling every second month from end of therapy until 2 years after diagnosis or hematological relapse. qPCR MRD data were available from at least 5 time points (median samples/patient: 13, range 5-19) after induction therapy in 42 patients; RUNX1-RUNX1T1 (8), CBFβ/MYH11 (4), MLLT3/MLL (7), MLL-ENL (2), and WT1 overexpression (21). WT1 was considered overexpressed if WT1/ABL ratio was higher than 1/16 at diagnosis. Patients with both fusion transcript and WT1 overexpression were monitored using the fusion transcript as MRD target.
Molecular remission (mCR) for fusion transcripts was defined as an MRD level in PB < 5x10-4. Molecular relapse was defined as a 10-fold increase in MRD to a level of at least 5x10-4.
A remission baseline WT1 expression was defined as the mean of the last three WT1 levels during consolidation therapy. Molecular relapse in patients with WT1 overexpression was defined as a 10-fold increase in MRD compared to baseline WT1 expression and above normal WT1 expression range.
Results: Median follow-up time in patients with fusion transcripts was 74 weeks from diagnosis (range: 20-169). Seven of 21 patients with fusion transcripts experienced molecular relapse during follow-up and all patients subsequently experienced hematological relapse. Median interval between molecular and hematological relapse was 3.4 weeks (range: 0.1-18.1). Patients in continuous mCR remained in CR1.
Median follow-up time in patients with WT1 overexpression was 116 weeks from diagnosis (range: 40-169). Five of 21 patients developed hematological relapse. In all cases relapse was preceded by molecular relapse. Median interval between molecular and hematological relapse was 2.7 weeks (range: 2.0-5.9). A similar increase in WT1 expression was not detected in any long-term CR1 patients.
Conclusion: Introduction of WT1 facilitates molecular disease monitoring in the vast majority of pediatric AML patients in CR1. Although numbers are small, our study indicates that both fusion transcripts and WT1 may serve as sensitive MRD targets. All patients in continuous CR1 maintained mCR throughout follow-up. All overt hematological relapses were preceded by molecular relapse though in 50% of cases the interval between molecular and hematological relapse was 3 weeks or less. Interval from molecular to hematological relapse showed great diversity in patients with fusion transcripts indicating heterogeneous relapse kinetics according to the underlying genetic lesion. A shorter interval between monitoring sampling may allow earlier detection of molecular relapse and a potential window for preemptive therapy.
No relevant conflicts of interest to declare.
Introduction. Chronic myeloid leukemia (CML) is a very rare disease in children and adolescents. The international registry of CML in children and adolescents (I-CML-Ped Study) established in the ...year 2011 has proven very beneficial to better describe this disease in pediatric cohorts.
Aims: To describe the characteristics and the outcome of the pediatric population with advanced phases of CML at initial diagnosis.
Patients and Methods: Patients enrolled in the international registry for CML in children and adolescents (I-CML-Ped Study) were the subjects of the study .Disease phases were defined according to the ELN criteria. Characteristics and outcome were determined in children in accelerated (AP) and blastic phase (BP). No recommendations were given regarding the treatment of the patients. Overall survival (OS) was estimated from diagnosis by the Kaplan and Meier method.
Results. 479 children and adolescents less than 18 years old with CML were enrolled in the I-CML-Ped study between January 2011 and April 2017. Among them, 37 patients (7.7%) presented at initial diagnosis with CML in advanced phase according to the ELN criteria: 20 children (13 boys and 7 girls, median age 12.6 years - range 3 to 18 years) and 17 children (11 boys and 6 girls, median age 11 years - range 5 to 17 years) were diagnosed in AP and in BP, respectively. Among the 17 patients diagnosed in BP, 12 (70%) had lymphoid and 2 myeloid phenotype (not specified: 2 patients with extramedullary disease as unique criteria of BP). According to the Sokal score for patients less than 45 years, 73% and 82% of the patients in AP and BP at diagnosis were high risk patients, respectively. For the 17 children in BP, treatment consisted of tyrosine kinase inhibitors (TKI) (n=3) or a combination of chemotherapy and TKI successively and/or simultaneously (n=14); 11 of them were transplanted (sibling donor: n=2; unrelated donor n=8; unknown: n=1) with a median time after diagnosis of 7 months (range, 4.5 to 10 months). For the 20 children in AP, treatment consisted of TKI (n=17) or a combination of chemotherapy and TKI successively and/or simultaneously (n=3); 6 of them were transplanted (sibling donor: n=5; unrelated donor n=1) with a median time after diagnosis of 8 months (range, 4 to 21.5 months). With a median time from diagnosis of 3.5 years (range 8 months to 13.5 years), 13 out of 17 patients in BP at initial diagnosis are alive in at least major molecular response (MMR) (11 patients) or without MMR (2 patients) including 5 patients who were not transplanted; death occurred in 3 transplanted patients (relapses: 2 patients; chronic graft versus host disease: 1 patient) and in one patient with progression of the disease. Nineteen out of 20 patients in AP at initial diagnosis are alive (one patient died after progression) in at least MMR (15 patients) or without MMR (2 patients) or unknown status (2 patients). The 5 years OS rates were 94% (95% confidence interval (CI): 66%-99%) and 74% (95% CI: 44%-89%) for patients diagnosed in AP or BP, respectively.
Conclusion: The data indicates that the frequency of advanced phases at diagnosis of CML is similar in children and in adults but with a predominance of lymphoid phenotype in children in blastic phase compared to adult population. Hematopoietic stem cell transplantation remains a curative treatment of children in blastic phase at diagnosis.
Acknowledgment: The I-CML Ped study is supported by an unrestricted grant from Novartis Pharmaceutical Company
No relevant conflicts of interest to declare.
Acute lymphoblastic leukemia (ALL) arising in infants less than 1 year of age is characterized by genetic rearrangements of the KMT2A gene (previously MLL) and an exceedingly poor prognosis. We have ...previously shown that infant KMT2A -rearranged (KMT2A -R) ALL has one of the lowest numbers of somatic mutations of any sequenced cancer with a mean of only 1.3 non-silent mutations being present in all leukemia cells per patient (Andersson et al., Nat Genet, 2017). Despite the paucity of mutations, activating mutations within the PI3K/RAS signaling pathway were present in about half of cases, most of which were subclonal with a mutant allele frequency (MAF) <0.30. In addition, some patients harbored multiple activating mutations at varying MAFs, suggesting the presence of multiple low-frequency leukemia clones. Based on these findings, we hypothesized that the genetic landscape of infant KMT2A -R ALL is more clonally heterogeneous than other pediatric leukemias, which might contribute to its poor prognosis as current treatment regimens may not eliminate all clones.
To test our hypothesis, high coverage bulk and single-cell genomics were used to study the mutation profiles of subclones in diagnostic samples from four infants younger than 6 months of age with KMT2A -R ALL. This included patients with t(4;11)(q21;q23) KMT2A-AFF1 (n=3) of which two relapsed and a patient with t(11;19)(q23;p13.3) KMT2A - MLLT1 (n=1) that remains in remission. DNA was isolated from bulk leukemia cells and high-depth paired-end whole-genome sequencing was performed on paired diagnostic and remission samples at an average haploid coverage of 160x and 35x, respectively. In addition, whole-exome sequencing was performed on the diagnostic samples at an average exon coverage of 375x. Somatic alterations, including single-nucleotide variations (SNVs), insertions-deletions (indels), structural variations (SVs), and copy number alterations, were detected using multiple analytical pipelines. In parallel, approximately 75 viable single cells from each diagnostic sample were isolated and the DNA from the cells underwent whole genome amplification using the Fluidigm C1 system.
Analyses of the whole-genome sequencing data from the four infant cases revealed an average of 715 putative sequence variants (range of 54-1199) per case across the genome. Using the combined data from whole-genome- and exome sequencing, an average of 55 (range 51-65) non-silent mutations affecting coding genes or non-coding RNAs were identified, with 83% having support by both sequencing methods, validating their presence. An average of 63% and 92% of the non-silent mutations detected by whole-genome and exome sequencing, respectively, were subclonal, demonstrating the enhanced ability to detect low frequency variants with higher sequencing depth.
Subclonal mutations in genes within the PI3K/RAS signaling pathway were detected in 2/4 cases with each patient harboring both a KRASG12D and a FLT3V491L as well as an additional NRASG13D for one of the cases. The recurrent FLT3V491L mutation is present in COSMIC and has been previously detected in leukemia, but to the best of our knowledge, it has not been experimentally verified to be activating.
We are now performing high-throughput amplicon-based sequencing at the single cells from those patients using 150 targets/cell to determine the number of distinct clones into which the bulk mutations segregate. The targets include a mixture of coding, intergenic variants and SVs. In addition, we are determining if each cell contains a mutation at a selected number of loci that are leukemia-associated mutational hotspots. After acquiring the data, we will reconstruct the clonal architecture of the samples, enabling us to assess the clonal heterogeneity and mutational histories of the leukemias.
Taken together, these data will provide a unique look into the underlying biology of infant KMT2A -R ALL by providing new details about the population genetic diversity and temporal genetic changes that occur during KMT2A -R infant leukemogenesis. These data may also provide insight into the universally poor prognosis of patients with KMT2A -R infant ALL.
No relevant conflicts of interest to declare.
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