Fms‐like tyrosine kinase 3 (
FLT
3
) internal tandem duplication (
ITD
) mutations, common in pediatric acute myeloid leukemia (
AML
), associate with early relapse and poor prognosis. Past studies ...have suggested additional cooperative mutations are required for leukemogenesis in
FLT
3
‐
ITD
+
AML
. Using
RNA
sequencing and a next‐generation targeted gene panel, we broadly characterize the co‐occurring genomic alterations in pediatric cytogenetically normal (
CN
)
FLT
3
‐
ITD
+
AML
to gain a deeper understanding of the clonal patterns and heterogeneity at diagnosis and relapse. We show that chimeric transcripts were present in 21 of 34 (62%) of
de novo
samples, 2 (6%) of these samples included a rare reoccurring fusion partner
BCL
11B
. At diagnosis, the median number of mutations other than
FLT
3 per patient was 1 (range 0–3), which involved 8 gene pathways;
WT
1
and
NPM
1
mutations were frequently observed (35% and 24%, respectively). Fusion transcripts and high variant allele frequency (
VAF
) mutants, which included
WT
1
,
NPM
1
,
SMARCA
2
,
RAD
21
, and
TYK
2
, were retained from diagnosis to relapse. We did observe reduction in
VAF
of simple or single mutation clones, but
VAF
s were preserved or expanded in more complex clones with multiple mutations. Our data provide the first insight into the genomic complexity of pediatric
CN
FLT
3
‐
ITD
+
AML
and could help stratify future targeted treatment strategies.
We compared the antitumor activities of the multitargeted tyrosine kinase inhibitors imatinib, sorafenib, and sunitinib to determine which inhibitor is best suited to be used for the treatment of ...acute myelogenous leukemia (AML). In nine human AML cell lines, sorafenib and sunitinib were more potent inhibitors of cellular proliferation than imatinib (IC50, 0.27 to >40, 0.002-9.1, and 0.007-13 micromol/L for imatinib, sorafenib, and sunitinib, respectively). Sorafenib and sunitinib were potent inhibitors of cells with fms-like tyrosine kinase 3 internal tandem duplication (IC50, 2 and 7 nmol/L) and c-KIT N822K mutations (IC50, 23 and 40 nmol/L). In four cell lines (MV4-11, Kasumi-1, KG-1, and U937) that spanned a range of drug sensitivities, sorafenib and sunitinib had similar activity in apoptosis and cell cycle assays, except that sunitinib did not promote apoptosis in U937 cells. Both drugs inhibited mitogen-activated protein kinase signaling but had no effect on AKT signaling in most of the cell lines tested. Sorafenib was substantially more bound than sunitinib in human plasma (unbound fraction, 0.59% versus 8.4%) and cell culture medium (unbound fraction, 1.3% versus 39%), indicating that sorafenib was more potent than sunitinib and that unbound sorafenib concentrations with activity against most AML cell lines are achievable in vivo. There was more intracellular accumulation of sorafenib than of sunitinib and imatinib in AML cells. Between 1 and 10 micromol/L, sorafenib inhibited the proliferation of six of nine primary AML blast samples by > or =50%. Our results highlight the pharmacologic features of sorafenib that may provide it an advantage in the treatment of AML.
Balanced rearrangements involving the KMT2A gene, located at 11q23, are among the most frequent chromosome aberrations in acute myeloid leukemia (AML). Because of numerous fusion partners, the ...mutational landscape and prognostic impact of specific 11q23/KMT2A rearrangements are not fully understood. We analyzed clinical features of 172 adults with AML and recurrent 11q23/KMT2A rearrangements, 141 of whom had outcome data available. We compared outcomes of these patients with outcomes of 1,097 patients without an 11q23/KMT2A rearrangement categorized according to the 2017 European LeukemiaNet (ELN) classification. Using targeted next-generation sequencing, we investigated the mutational status of 81 leukemia/cancer-associated genes in 96 patients with 11q23/KMT2A rearrangements with material for molecular studies available. Patients with 11q23/KMT2A rearrangements had a low number of additional gene mutations (median, 1; range 0 to 6), which involved the RAS pathway (KRAS, NRAS, and PTPN11) in 32% of patients. KRAS mutations occurred more often in patients with t(6;11)(q27;q23)/KMT2A-AFDN compared with patients with the other 11q23/KMT2A subsets. Specific gene mutations were too infrequent in patients with specific 11q23/KMT2A rearrangements to assess their associations with outcomes. We demonstrate that younger (age 60 y) patients with t(9;11)(p22;q23)/KMT2A-MLLT3 had better outcomes than patients with other 11q23/KMT2A rearrangements and those without 11q23/KMT2A rearrangements classified in the 2017 ELN intermediate-risk group. Conversely, outcomes of older patients (age ≥60 y) with t(9;11)(p22;q23) were poor and comparable to those of the ELN adverse-risk group patients. Our study shows that patients with an 11q23/KMT2A rearrangement have distinct mutational patterns and outcomes depending on the fusion partner.
Introduction: Acute Myeloid Leukemia (AML) is a genetically heterogeneous disease, associated with mutations in a well-established set of genes that affect myeloid cell differentiation and ...proliferation. While treatment options were limited for many decades, many new pharmacological treatments in the form of small molecule inhibitors have been developed in recent years. In order to be effective, many of these inhibitors require the presence of specific biomarkers that must be detected with appropriate genetic screening assays. Genetic studies may also inform conventional care plans. The cost and time of genetic testing can be limiting factor, delaying the start of treatment and often requiring referral to larger regional medical center. Such delays can result in increased morbidities and mortality in this patient population, making faster and more accessible genetic testing highly relevant.
Methods: We have developed a rapid single-molecule sequencing-based assay which requires minimal laboratory equipment, while providing same-day results. We utilized amplification-based library preparation to generate sequencing libraries for the Oxford Nanopore Technologies MinION instrument. We have combined rapid library preparation, real-time single-molecule sequencing, and novel computational analysis algorithms to rapidly screen patient blood samples for the presence of actionable biomarkers. In this study, we analyzed samples from 48 AML patients with known mutational statuses to evaluate the performance of our approach. For our proof of principle assay, we included seven molecular targets (six hotspot mutations; one duplication) in five AML-associated genes (DNMT3A, FLT3, IDH1, IDH2, and JAK2). We compared the accuracy of our assay to current gold standard approaches which include Illumina-based short-read sequencing and capillary electrophoresis.
Results and Discussion: Our assay exhibited equal or superior accuracy compared to the current gold standard approaches, while providing results significantly faster (in less than 6 hours). Each of the 48 samples were correctly classified by mutational status, with the detected variant allele frequency exhibiting high correlation (R2 for mutational hotspots ranging from a low of 0.84 to a high of 0.99) between our Nanopore-based assay and the control Illumina-based assay. Our custom FLT3-ITD detection algorithm correctly identified 24/24 samples as ITD positive, with insertion lengths matching expected peak lengths from capillary electropherogram testing and highly concordant allelic ratios. In contrast, an assay using Illumina sequencing data correctly detected only 6/24 ITD positive samples. Therefore, we conclude that our assay provides superior variant detection accuracy, significantly shorter time to results (4-6 hours instead of days), and at an expected cost of below $250/sample when utilizing single-sample Flongle flow cells. Critically, this procedure is accessible beyond tertiary care academic medical centers and could be deployed at the point-of-care in smaller hospitals, physician offices, and resource-constrained settings worldwide.
Display omitted
No relevant conflicts of interest to declare.
Thus far, only 5-15% of AML patients aged ≥60 years are cured with chemotherapy. Identification of patients who are less (more) likely to respond to standard chemotherapy might enable early risk ...stratification toward alternative treatment regimens. We used a next-generation sequencing panel of 80 cancer- and/or leukemia-associated genes to profile molecularly 423 older patients with de novo AML. Using variables identified in multivariable models and co-occurring mutations in NPM1-mutated AML, we classified the patients into good-, intermediate-, and poor-risk groups for complete remission (CR) attainment, disease-free (DFS), and overall survival (OS). Whereas 81% of good-risk patients (comprising NPM1-mutated patients harboring mutations in chromatin remodeling, cohesin complex, methylation-related, spliceosome, and/or RAS pathway genes, FLT3-TKD, and/or patients without FLT3-ITD) achieved a CR, only 32% of poor-risk patients (with U2AF1, WT1 mutations and/or complex karyotype) did. Intermediate-risk patients had a 50% CR rate. Similarly, using NPM1 co-mutation patterns and SF1 mutation status, we identified patients with favorable DFS and OS 3-year rates of 46% and 45%, respectively. Patients with adverse genetic features had DFS and OS rates of only 2% and 4%. We show that application of our proposed criteria may refine the 2017 European LeukemiaNet classification for older patients treated with chemotherapy.
Core-binding factor acute myeloid leukemia (CBF-AML) is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion ...genes require a 'second hit' to initiate leukemogenesis. Mutation assessment of 177 adults with CBF-AML, including 68 with t(8;21) and 109 with inv(16)/t(16;16), identified not only mutations well known in CBF-AML but also mutations in the CCND1 and CCND2 genes, which represent novel frequent molecular alterations in AML with t(8;21). Altogether, CCND1 (n=2) and CCND2 (n=8) mutations were detected in 10 (15%) patients with t(8;21) in our cohort. A single CCND2 mutation was also found in 1 (0.9%) patient with inv(16). In contrast, CCND1 and CCND2 mutations were detected in only 11 (0.77%) of 1426 non-CBF-AML patients. All CCND2 mutations cluster around the highly conserved amino-acid residue threonine 280 (Thr280). We show that Thr280Ala-mutated CCND2 leads to increased phosphorylation of the retinoblastoma protein, thereby causing significant cell cycle changes and increased proliferation of AML cell lines. The identification of CCND1 and CCND2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease.
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