Project description:Acute myeloid leukemia (AML) is caused by genetic aberrations that also govern the prognosis of patients and guide risk-adapted and targeted therapy. Genetic aberrations in AML are structurally diverse and currently detected by different diagnostic assays. This study sought to establish whole transcriptome RNA sequencing as single, comprehensive, and flexible platform for AML diagnostics. We developed HAMLET (Human AML Expedited Transcriptomics) as bioinformatics pipeline for simultaneous detection of fusion genes, small variants, tandem duplications, and gene expression with all information assembled in an annotated, user-friendly output file. Whole transcriptome RNA sequencing was performed on 100 AML cases and HAMLET results were validated by reference assays and targeted resequencing. The data showed that HAMLET accurately detected all fusion genes and overexpression of EVI1 irrespective of 3q26 aberrations. In addition, small variants in 13 genes that are often mutated in AML were called with 99.2% sensitivity and 100% specificity, and tandem duplications in FLT3 and KMT2A were detected by a novel algorithm based on soft-clipped reads with 100% sensitivity and 97.1% specificity. In conclusion, HAMLET has the potential to provide accurate comprehensive diagnostic information relevant for AML classification, risk assessment and targeted therapy on a single technology platform.
Project description:BackgroundAcute myeloid leukemia (AML) is characterized by uncontrolled proliferation of malignant hematopoietic cells in the bone marrow that are arrested in differentiation. In AML pathogenesis, hematopoietic progenitor cells acquire multiple genetic aberrations often occurring in the same set of genes that ultimately lead to malignant transformation. In the WHO classification 2016, six AML classes with different prognosis are identified by chromosomal translocations measured by standard cytogenetics. All balanced translocations produce fusion genes, except for t(3;3)/inv(3;3), which lead to overexpression of MECOM/EVI1 associated with poor prognosis. For accurate risk assessment of cytogenetically normal AML, four genes need to be screened to distinguish AML with mutated NPM1 in the absence of FLT3 mutations and AML with bi-allelic CEBPA mutations, which have a favorable prognosis, and AML with RUNX1 mutations which have a poor prognosis. Recently, a full genomic classification system has been proposed by Papaemmanuil et al. (NEJM 2016). In this system, the same six classes with chromosomal translocations are identified by standard cytogenetics as well as five additional classes characterized by genetic mutations in 14 different genes. AimThe aim of the study was to investigate whether whole transcriptome RNA-sequencing (RNAseq) can be used as single technology for classification of AML. MethodA panel of 100 AML were analyzed and a bio-informatics pipeline called HAMLET (Human AML Expedited Transcriptomics) was developed in which 4 modules are integrated to detect (1) fusion genes, (2) small variants in 13 recurrently mutated genes, (3) internal tandem duplications in FLT3 (FLT3-ITD) and partial tandem duplications in KMT2A (KMT2A-PTD) and (4) overexpression of MECOM/EVI1. All mutations that were called by HAMLET were validated by diagnostic data as available for all 100 AML or targeted PCR followed by Sanger or next generation sequencing on all positive AML and at least an equal number of negative cases. Results & discussionThe data showed that HAMLET accurately identified all genetic aberrations with high sensitivity and specificity. In addition, in 7 AML, fusion transcripts were detected that are not measured by standard cytogenetics including three cases that lack any class-defining lesion according to Papaemmanuil et al. Moreover, overexpression of MECOM/EVI1 was detected in two AML with inv(3) as well as in five cases without inv(3)/t(3;3), and a gene signature was measured to distinguish AML with CEBPA mutations with favorable prognosis. In conclusion, HAMLET provides a comprehensive and flexible pipeline for RNAseq analysis to retrieve all relevant information for current classification of AML as well as additional information that may improve classification in the future.
Project description:Pediatric AML is characterized by numerous genetic aberrations (chromosomal translocations, deletions, insertions) impacting its classification for risk of treatment failure. Aberrations are described by classical cytogenetic procedures (karyotyping, FISH), which harbor limitations (low resolution, need for cell cultivation, cost-intensiveness, experienced staff required). Optical Genome Mapping (OGM) is an emerging chip-based DNA technique combining high resolution (~500 bp) with a relatively short turnaround time. Twenty-four pediatric patients with AML, bi-lineage leukemia, and mixed-phenotype acute leukemia were analyzed by OGM, and the results were compared with cytogenetics. Results were discrepant in 17/24 (70%) cases, including 32 previously unknown alterations called by OGM only. One newly detected deletion and two translocations were validated by primer walking, breakpoint-spanning PCR, and DNA sequencing. As an added benefit, in two cases, OGM identified a new minimal residual disease (MRD) marker. Comparing impact on risk stratification in de novo AML, 19/20 (95%) cases had concordant results while only OGM unraveled another high-risk aberration. Thus, OGM considerably expands the methodological spectrum to optimize the diagnosis of pediatric AML via the identification of new aberrations. Results will contribute to a better understanding of leukemogenesis in pediatric AML. In addition, aberrations identified by OGM may provide markers for MRD monitoring.
Project description:Recent advances in the genetic understanding of acute myeloid leukemia (AML) have improved clinical outcomes in pediatric patients. However, ∼40% of patients with pediatric AML relapse, resulting in a relatively low overall survival rate of ∼70%. The objective of this study was to reveal the comprehensive genetic background of pediatric AML. We performed transcriptome analysis (RNA sequencing [RNA-seq]) in 139 of the 369 patients with de novo pediatric AML who were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 trial and investigated correlations between genetic aberrations and clinical information. Using RNA-seq, we identified 54 in-frame gene fusions and 1 RUNX1 out-of-frame fusion in 53 of 139 patients. Moreover, we found at least 258 gene fusions in 369 patients (70%) through reverse transcription polymerase chain reaction and RNA-seq. Five gene rearrangements were newly identified, namely, NPM1-CCDC28A, TRIP12-NPM1, MLLT10-DNAJC1, TBL1XR1-RARB, and RUNX1-FNBP1. In addition, we found rare gene rearrangements, namely, MYB-GATA1, NPM1-MLF1, ETV6-NCOA2, ETV6-MECOM, ETV6-CTNNB1, RUNX1-PRDM16, RUNX1-CBFA2T2, and RUNX1-CBFA2T3. Among the remaining 111 patients, KMT2A-PTD, biallelic CEBPA, and NPM1 gene mutations were found in 11, 23, and 17 patients, respectively. These mutations were completely mutually exclusive with any gene fusions. RNA-seq unmasked the complexity of gene rearrangements and mutations in pediatric AML. We identified potentially disease-causing alterations in nearly all patients with AML, including novel gene fusions. Our results indicated that a subset of patients with pediatric AML represent a distinct entity that may be discriminated from their adult counterparts. Based on these results, risk stratification should be reconsidered.
Project description:Nucleosides and their analogues constitute an essential family of anticancer drugs. DNA has been the presumptive target of the front-line prodrug for acute myeloid leukemia (AML), cytarabine (ara-C), since the 1980s. Here, the biomolecular targeting of ara-C was evaluated in primary white blood cells using the ara-C mimic "AzC" and azide-alkyne "click" reactions. Fluorescent staining and microscopy revealed that metabolic incorporation of AzC into primary white blood cells was unexpectedly enhanced by the DNA polymerase inhibitor aphidicholine. According to RNaseH digestion and pull-down-and-release experiments, AzC was incorporated into short RNA fragments bound to DNA in peripheral blood monocytes (PBMCs) collected from all six healthy human donors tested. Samples from 22 AML patients (French-American-British classes M4 and M5) exhibited much more heterogeneity, with 27% incorporating AzC into RNA and 55% into DNA. The overall survival of AML patients whose samples incorporated AzC into RNA was approximately 3-fold higher as compared to that of the DNA cohort (p ≤ 0.056, χ2 = 3.65). These results suggest that the RNA primers of DNA synthesis are clinically favorable targets of ara-C, and that variable incorporation of nucleoside drugs into DNA versus RNA may enable future patient stratification into treatment-specific subgroups.
Project description:Acute myeloid leukemia (AML) is an aggressive hematological tumor caused by the malignant transformation of myeloid progenitor cells. Although intensive chemotherapy leads to an initial therapeutic response, relapse due to drug resistance remains a significant challenge. In recent years, accumulating evidence has suggested that post-transcriptional methylation modifications are strongly associated with tumorigenesis. However, the mRNA profile of m7G modification in AML and its role in drug-resistant AML are unknown. In this study, we used MeRIP-seq technology to establish the first transcriptome-wide m7G methylome profile for AML and drug-resistant AML cells, and differences in m7G between the two groups were analyzed. In addition, bioinformatics analysis was conducted to explore the function of m7G-specific methylated transcripts. We found significant differences in m7G mRNA modification between AML and drug-resistant AML cells. Furthermore, bioinformatics analysis revealed that differential m7G-modified mRNAs were associated with a wide range of cellular functions. Importantly, down-methylated m7G modification was significantly enriched in ABC transporter-related mRNAs, which are widely recognized to play a key role in multidrug resistance. Our results provide new insights into a novel function of m7G methylation in drug resistance progression of AML.
Project description:Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with an incidence of over 20?000 cases per year in the United States alone. Large chromosomal translocations as well as mutations in the genes involved in hematopoietic proliferation and differentiation result in the accumulation of poorly differentiated myeloid cells. AML is a highly heterogeneous disease; although cases can be stratified into favorable, intermediate and adverse-risk groups based on their cytogenetic profile, prognosis within these categories varies widely. The identification of recurrent genetic mutations, such as FLT3-ITD, NMP1 and CEBPA, has helped refine individual prognosis and guide management. Despite advances in supportive care, the backbone of therapy remains a combination of cytarabine- and anthracycline-based regimens with allogeneic stem cell transplantation for eligible candidates. Elderly patients are often unable to tolerate such regimens, and carry a particularly poor prognosis. Here, we review the major recent advances in the treatment of AML.
Project description:Acute myeloid leukemia (AML) cells have high oxidative phosphorylation and mitochondrial mass and low respiratory chain spare reserve capacity. We reasoned that targeting the mitochondrial RNA polymerase (POLRMT), which indirectly controls oxidative phosphorylation, represents a therapeutic strategy for AML. POLRMT-knockdown OCI-AML2 cells exhibited decreased mitochondrial gene expression, decreased levels of assembled complex I, decreased levels of mitochondrially-encoded Cox-II and decreased oxidative phosphorylation. POLRMT-knockdown cells exhibited an increase in complex II of the electron transport chain, a complex comprised entirely of subunits encoded by nuclear genes, and POLRMT-knockdown cells were resistant to a complex II inhibitor theonyltrifluoroacetone. POLRMT-knockdown cells showed a prominent increase in cell death. Treatment of OCI-AML2 cells with 10-50 µM 2-C-methyladenosine (2-CM), a chain terminator of mitochondrial transcription, reduced mitochondrial gene expression and oxidative phosphorylation, and increased cell death in a concentration-dependent manner. Treatment of normal human hematopoietic cells with 2-CM at concentrations of up to 100 µMdid not alter clonogenic growth, suggesting a therapeutic window. In an OCI-AML2 xenograft model, treatment with 2-CM (70 mg/kg, i.p., daily) decreased the volume and mass of tumours to half that of vehicle controls. 2-CM did not cause toxicity to major organs. Overall, our results in a preclinical model contribute to the functional validation of the utility of targeting the mitochondrial RNA polymerase as a therapeutic strategy for AML.
Project description:Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML.