Clonal Evolution Of Acute Myeloid Leukemia Research Articles

Clonal Evolution in 207 Cases of Refractory or Relapsed Acute Myeloid Leukemia.

Clonal evolution (CE) is a driving force behind the development and progression of acute myeloid leukemia (AML). Advances in molecular and cytogenetic assays have improved the depth and breadth of detection of CE in AML, which is defined here as a detected change in cytogenetic or molecular profile at relapsed or refractory (RR) disease. In this study, we demonstrate the clinical impact of CE in a cohort of patients with RR AML treated between 2013 and 2023. We discovered CE is significantly more frequent in relapsed disease (58.2%, [46.6%, 69.2%]) than in refractory disease (21.1%, [14.4%, 29.2%], p < 0.001). CE negatively impacts prognosis when detected by conventional karyotyping in refractory disease (4.2 vs. 13.9 months, p < 0.011). In contrast with prior literature, CE had no impact on overall survival if detected in relapsed disease. Surprisingly, those who achieved negative measurable residual disease (MRD) were no more likely to eliminate their original clone than those who did not (p = 1). We found several cytogenetic and molecular signatures which may predispose to CE: aberrations of chromosome 17, trisomy 8, TP53, KRAS, and FLT3-TKD. Finally, physicians were less likely to retreat those with CE with IC after receiving IC as first-line therapy (35.0% vs. 70.9%, p = 0.004). This study illustrates the role of CE in chemotherapy-resistant AML; we identify unique cytogenetic and molecular signatures that define a subset of patients associated with a dismal prognosis. As next-generation sequencing panels expand and new methods to characterize cytogenetic abnormalities emerge, our findings establish a basis for future studies investigating the prognostic and therapeutic impact of CE.

Abstract A49: Characterizing the order of mutation acquisition in acute myeloid leukemia using large-scale single-cell sequencing

Abstract Introduction: Acute myeloid leukemia (AML) has a poor prognosis, despite aggressive therapies and a recent expansion in the array of treatments. Treatment is often determined by mutations, which risk-stratify a patient’s leukemia or can identify mutations that serve as therapeutic targets. Although common mutations in AML have been extensively studied, less research has formally characterized the order by which mutations tend to occur and how this order relates to properties of the disease. Methods: We leveraged published, single-cell DNA sequencing data from three institutions to model the clonal evolution of AML. Sequencing had been performed using targeted sequencing panels covering 19 to 37 genes, and driver mutations were identified using conservative filters. Mutation trees were created using Single Cell Inference of Tumor Evolution (SCITE). Clonal evolution patterns, including across clinical timepoints, were identified and compared to patient characteristics, disease phenotype, and outcomes. The BeatAML dataset was leveraged to explore associations between mutation order and gene expression. Results: Using 835 driver mutations from 276 samples and 209 patients, we identified 223 total unique mutation patterns. Branched evolution primarily involves FLT3 and RAS pathway mutations, whereas certain mutation pairs, such as NPM1 and FLT3, always occurred linearly in the same clone. Although some mutation pairs, such as those related to DNA methylation versus the RAS pathway, tended to occur in a specific order, several cases exhibited atypical orderings. Early signaling gene mutations were associated with younger patient age and increased signaling mutation homozygosity while NRAS-first cases were associated with increased monocyte counts. NRAS-first cases were also associated with distinct gene expression patterns in the BeatAML dataset. Paired diagnosis and relapse samples revealed novel associations between mutations gained at relapse and their clonal context, and these analyses supported a relative fitness advantage for signaling mutations in clones containing mutations in NPM1 or genes affecting DNA methylation. Conclusions: Clonal evolution in AML can be reconstructed at scale using single-cell DNA sequencing data, and different mutation acquisition patterns are associated with distinct leukemia and patient characteristics, despite cases having similar co-mutation patterns. Citation Format: Matthew Schwede, Katharina Jahn, Linde A Miles, Robert L Bowman, Jack Kuipers, Troy M Robinson, Asiri Ediriwickrema, Andrew J Gentles, Ross Levine, Niko Beerenwinkel, Koichi Takahashi, Ravindra Majeti. Characterizing the order of mutation acquisition in acute myeloid leukemia using large-scale single-cell sequencing [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A49.

Molecular and Clinical PHF6 Mutant Myeloid Neoplasia Provides Clues As to Their Pathogenesis and Therapeutic Targeting

Congenital PHF6 mutations (PHF6MT) cause the X-linked neurodevelopmental disorder, Börjeson-Forssman-Lehmann syndrome (BFLS). In contrast, acquired PHF6MT are frequently detected in hematologic neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and T-cell acute lymphoblastic leukemia (T-ALL). Indeed, PHF6 is highly expressed in brain/developing CNS as well as in hematopoietic subpopulations suggesting its role in neurogenesis and hematopoiesis consistent with the neurologic BFSL presentation of the germline and leukemias with somatic mutations of this gene. This study focuses on involvement of PHF6 lesions in AML. Here, we hypothesize that there are functional links between lymphoid and myeloid neoplasia (MN) with PHF6-deficiency that can be instructive in terms of the impact of this gene as a tumor suppressor gene (TSG). In total, we analyzed sequencing results of 6886 AML cases, including 616 cases from open sources such as The Beat AML Master dataset (Tyner JW , Tognon CE, Bottomly D, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562(7728):526-531). We first investigated the clinical phenotype associated with PHF6MT. We detected a total of 111 (1.6% of cases) PHF6MT. While frameshifts were found across all the coding regions, missense and stop-gains were concentrated in the two PHD-type zinc finger domains and were overlapping between other MN and the previously reported T-ALL cases. PHF6MT were significantly more common in male cases (2.3 vs. 0.8%; p<.001) similar to other X-genes such as UBA1, ZRSR2, and STAG2 but different from ATRX, PIGA, and KDM6A (UTX). When comparing primary AML (pAML) with secondary AML (sAML), sAML had more PHF6MT than pAML (2.9 vs. 1.4%; p<.001). While PHF6MT were not associated with chromosomal abnormalities in male cases, female cases with X chromosome deletion (delX) showed higher frequency of PHF6MT compared to females with diploid X chromosome (6.5 vs. 0.8%; p=.0019). Consequently, when delX were included, a total of 2.3% of female patients had PHF6 hits with 10% of them in a homo/hemizygous configuration. While RUNX1, U2AF1, and ASXL1 mutations coincided with PHF6 hits in males, in female cases, delX and ASXL1 mutations were the most common associated defects (Fig.1). Interestingly, RUNX1 mutations (RUNX1MT) in females were enriched in cases with delX. As a result, PHF6MT were enriched into two distinct subclasses: while PHF6MT were enriched in male cases with sAML and RUNX1MT (8.8%), all female cases with sAML, RUNX1MT, and delX also acquired PHF6MT (Fig.2). A higher frequency of PHF6MT in advanced MN than early diseases (e.g., low-risk MDS), suggestive of a secondary rather than a founder role of PHF6MT prompted us to study the clonal architecture of PHF6MT cases using variant allelic frequencies. RUNX1MT were dominant/co-dominant to PHF6 hits in the clonal evolution of AML in contrast to T-ALL, in which PHF6MT appears to be an earlier event. Similar relationship was found with regard to PHF6 hits and other myeloid gene mutations (e.g., ASXL1, DNMT3A, or SRSF2) suggesting a lineage-restricted TSG function of PHF6 in AML following myeloid commitment (consistent with the physiologic expression of PHF6 in pre-B, pre-T, and erythroid progenitors during hematopoietic ontogeny). PHF6 mutations also affected AML patients’ outcome. Overall, PHF6MT showed worse outcome in male cases, but not in female cases. However, if PHF6MT cases also had RUNX1 mutations, the outcome was extremely low in both sexes. Unsupervised RNAseq analysis of PHF6 mutational status vs. expression pattern revealed that PHF6MT cases showed higher mRNA levels of several lymphoid genes (GCSAM, LY9, or BLNK). This finding was also confirmed by RNAseq of in-house PHF6 knock-out K562 and U-937 clones. Immunohistochemistry unveiled that LY9 was positive in illustrative PHF6MT cases and flow cytometry revealed the presence of lymphoid markers in 2/3 PHF6MT cases. Our study confirms that PHF6MT are more common in male AML. Also we demonstrate the intricate relationships between PHF6MT and RUNX1MT in male sAML patients which extend also to female cases with delX. In AML, PHF6MT are a late event compared to T-ALL, in which they occur early in leukemogenesis. This is also in line with higher frequency of PHF6MT in sAML. Some PHF6MT cases have higher expression of lymphoid genes, which may be candidate for molecular targeted therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Integrated Multidimensional Flow Cytometry (MFC) and Next-Generation Sequencing (NGS) to Reconstruct Evolutionary Paterns from Dysplasia to Acute Myeloid Leukemia (AML)

Integrated Multidimensional Flow Cytometry (MFC) and Next-Generation Sequencing (NGS) to Reconstruct Evolutionary Paterns from Dysplasia to Acute Myeloid Leukemia (AML)

Downregulation of MHC Class II in Relapsed AML Cells after Allogeneic Transplantation

The evolutionary model of Acute Myeloid Leukemia (AML) progression suggests that malignant clones survive initial therapy and acquire new genetic and epigenetic changes, ultimately resulting in therapy-resistant disease. Allogeneic hematopoietic stem cell transplant (HCT) is a potent therapy that provides benefit in part through an immune-mediated Graft-vs-Leukemia (GVL) effect, although relapse after HCT remains a major clinical problem. Recent studies have demonstrated the gain and loss of specific AML-associated mutations at the time of relapse, consistent with a model of clonal evolution; however, a comprehensive screen of genetic changes in AML relapsed after HCT has not yet been reported. We hypothesized that the distinctive, immune-mediated GVL effect imposed by HCT may result in distinct patterns of tumor evolution in relapse after this therapy.To test this, we performed enhanced exome sequencing of matched diagnosis and relapse tumors to identify somatic mutations in 15 patients who relapsed after matched related or unrelated donor HSCT. For comparison, we analyzed 20 cases of AML relapsed after chemotherapy alone. In the majority of samples, new somatic variants were identified, and copy number analysis demonstrated gain and loss of genomic regions, as has been previously reported. However, compared to cases relapsed after chemotherapy alone, there were no gene mutations or copy number changes that were specific to relapse after HCT. Specifically, we found no changes in genes involved in antigen presentation, cytokine production, or immune checkpoints, and an unbiased pathway analysis revealed no enrichment for mutations in immune response pathways. Overall, the spectrum of mutations found at relapse after HCT did not differ from that observed in relapse after chemotherapy only.Since no canonical genetic changes were observed in relapsing AML after HCT, we speculated that clonal evolution of AML after HCT may instead occur through selection of randomly occurring epigenetic changes in AML cells that allow them to escape immune surveillance. To test this hypothesis, we performed RNA sequencing on purified AML blasts (CD45 positive, side scatter low) from presentation and relapsed AML sample pairs from both post-HSCT and post-chemo relapses. Interestingly, in the post chemotherapy relapse cases, only 8 genes were identified with expression changes that met pre-specified cutoffs for significance (FDR<0.5). In contrast, in the post relapse cohort, 34 genes were found to be significantly upregulated, and 187 genes were significantly downregulated, compared to the same sample at presentation. Gene pathway analysis revealed significant dysregulation of immune response genes (the most dysregulated pathway, p<10-35) as well as pathways encompassing cell adhesion/motility and the innate immune response.Four classical MHC Class II genes were among the 187 genes that were downregulated after post-HCT relapse: HLA-DPA1 (mean fold change -11.9 in 6/7 cases), HLA-DPB1 (mean fold change -6.5 in 6/7 cases), HLA-DQB1 (mean fold change -9.7 in 6/7 cases), and HLA-DRB1 (mean fold change -11.9 in 6/7 cases) (Figure 1). In these same 6 cases, there was a strong (but not significant) trend towards downregulation in the remaining MHC Class II genes: HLA-DQA1, HLA-DRB3, and HLA-DRA, as well as CIITA, a transcriptional activator believed to act as the “master regulator” of MHC Class II genes (Figure 1). Several other genes involved in MHC Class II antigen processing and presentation were significantly downregulated post-HCT (FDR<.05), including HLA-DMA, HLA-DMB, and CD74 . In contrast, there was no significant downregulation of MHC Class I genes observed after allo-relapse, raising the possibility that loss of MHC Class II presentation may play a particularly important role in immune evasion after HCT. To validate these findings, we performed flow cytometry for Class II and Class I genes on banked post-HCT relapse samples, and this confirmed significant downregulation of MHC Class II (but not Class I) expression on post-HCT relapse samples (Figure 2). Taken together, these results suggest that loss of MHC Class II-mediated antigen presentation may be an important mechanism of immune escape after HCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical significance of MRD in AML

Some studies have reported the clinical significance of minimal/measurable residual disease (MRD) in considering the prognostic stratification and therapeutic intervention after complete remission in acute myeloid leukemia (AML). In the clinical setting, multicolor flow cytometry (MFC), a quantitative PCR method targeting the expression of fusion genes generated by chromosomal translocation, such as PML-RARA, RUNX1-RUNXT1, and CBFB-MYH11, as well as WT1 mRNA, was used to detect MRD in AML. In recent years, quantitative PCR, next-generation sequence, and digital-droplet PCR methods targeting genetic alterations often detected in AML have been developed to assess its clinical significance. However, besides analysis methods, many common problems persist in MRD evaluation, such as sample collection points, type of samples, and threshold setting. Although several gene mutations involved in clonal hematopoiesis have been detected in CR patients, their presence did not correlate with the prognosis, and some leukemia-specific mutations did not always persist during the clonal evolution of AML. Therefore, it is essential to combine multiple methods, such as target gene mutation, quantitative PCR, and MFC to enhance the sensitivity of measurement. Furthermore, the establishment of novel treatment strategies incorporating MRD and molecular abnormalities is warranted for better clinical outcomes of AML.

Clonal evolution of acute myeloid leukemia from diagnosis to relapse.

Based on the individual genetic profile, acute myeloid leukemia (AML) patients are classified into clinically meaningful molecular subtypes. However, the mutational profile within these groups is highly heterogeneous and multiple AML subclones may exist in a single patient in parallel. Distinct alterations of single cells may be key factors in providing the fitness to survive in this highly competitive environment. Although the majority of AML patients initially respond to induction chemotherapy and achieve a complete remission, most patients will eventually relapse. These points toward an evolutionary process transforming treatment‐sensitive cells into treatment‐resistant cells. As described by Charles Darwin, evolution by natural selection is the selection of individuals that are optimally adapted to their environment, based on the random acquisition of heritable changes. By changing their mutational profile, AML cell populations are able to adapt to the new environment defined by chemotherapy treatment, ultimately leading to cell survival and regrowth. In this review, we will summarize the current knowledge about clonal evolution in AML, describe different models of clonal evolution, and provide the methodological background that allows the detection of clonal evolution in individual AML patients. During the last years, numerous studies have focused on delineating the molecular patterns that are associated with AML relapse, each focusing on a particular genetic subgroup of AML. Finally, we will review the results of these studies in the light of Darwinian evolution and discuss open questions regarding the molecular background of relapse development.

Abstract 2725: The single-cell atlas of driver mutations in acute myeloid leukemia

Abstract Assessment of intratumor genetic heterogeneity by next-generation sequencing (NGS) is confounded by tumor purity and zygosity of mutations. Furthermore, bulk NGS cannot visualize the cell-level co-occurrence or exclusivity among multiple mutations, causing the inaccurate inference of subclonal architecture. Here, we performed single cell DNA sequencing (scDNA seq) in 82 bone marrow samples from 70 patients with acute myeloid leukemia (AML) using a novel microfluidics-based platform covering 40 amplicons in 19 AML genes (Tapestri, Mission Bio). All samples were concurrently sequenced by the bulk NGS using 295 gene exome capture sequencing. Selected cases were also analyzed by SNP array (Illuminia Omni 2.5 array) to obtain allele-specific copy number data. In total, 319,406 cells (median 3,755 cells/sample) were genotyped with median allele drop-out rate of 8.7% (population frequency inferred from commonly heterozygous SNP loci). Each amplicon was covered at a median 24x/cell. The scDNA seq detected 230 of 238 (97%) bulk NGS-confirmed mutations. RUNX1 mutations were frequently detected as homozygous mutations and concurrent SNP array analysis detected copy number neutral loss-of-heterozygosity of RUNX1 in these cases. Additionally, in cases with homozygous FLT3-ITD mutations, SNP array detected allele-specific copy number gain of mutant loci, which likely resulted in homozygous calls. scDNA seq data unambiguously resolved the single-cell level co-occurrence of driver mutations in AML such as DNMT3A/FLT3-ITD/NPM1, SRSF2/IDH2, and ASXL1/RUNX1, confirming the cooperative function of these driver mutations. On the other hand, the data also revealed mutually exclusive relationships at cellular level between IDH1/IDH2, FLT3-ITD/TKD, NRAS/KRAS, NRAS/PTPN11, and SRSF2/EZH2 mutations, which supports the functional redundancy of these genes in leukemogenesis. Inference of phylogenetic trees using SCITE algorithm (Jahn et al. Genome Biology 2016) uncovered distinct patterns of clonal evolution in AML. The majority of the analyzed cases had a linear evolution pattern where the founder mutations linearly acquired sub-clonal mutations in a step-wise manner. We also detected convergent evolution in some cases where functionally similar driver mutations were acquired in parallel. DNMT3A, IDH1, IDH2 and U2AF1 mutations were frequently detected as trunk mutations, whereas FLT3, NRAS, and NPM1 mutations were usually detected as branch mutations. Analysis of longitudinal samples from 11 patients revealed the remodeling of clonal architecture at relapse. Patients who had 2 or more major subclones at baseline had significantly worse overall survival than those with one subclone (2-year OS 13 vs. 70 months, p = 0.0493). For the first time our data provides a comprehensive landscape of driver mutations and detailed clonal evolution history in AML at the single-cell level using high-throughput scDNA seq genotype information. Citation Format: Kiyomi Morita, Feng Wang, Katharina Jahn, Yuanqing Yan, Robert Durruthy-Durruthy, Anup Parikh, Jairo Matthews, Latasha Little, Curtis Gumbs, Jianhua Zhang, Xingzhi Song, Erika Thompson, Keyur Patel, Carlos Bueso-Ramos, Courtney DiNardo, Farhad Ravandi, Elias Jabbour, Michael Andreeff, Jorge Cortes, Marina Konopleva, Guillermo Garcia-Manero, Hagop Kantarjian, Dennis J. Eastburn, P Andrew Futreal, Niko Beerenwinkel, Koichi Takahashi. The single-cell atlas of driver mutations in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2725.

S833 THE SINGLE‐CELL ATLAS OF DRIVER MUTATIONS IN AML

Background:Assessment of genetic intratumor heterogeneity using next generation sequencing (NGS) can underestimate the complexity of subclonal architecture, since it is confounded by tumor purity, zygosity, and cell‐level co‐occurrence and exclusivity among multiple mutations.Aims:To thoroughly dissect the subclonal architecture, we performed single cell DNA sequencing (scDNA‐seq) in 98 bone marrow samples from 80 patients with acute myeloid leukemia (AML).Methods:We used a novel microfluidics‐based scDNA‐seq platform covering 40 amplicons in 19 AML genes (Tapestri, Mission Bio). As a reference, all samples were concurrently sequenced by the bulk NGS using 295‐gene exome capture sequencing. Allele‐specific copy number data was obtained from SNP array data (Illuminia Omni 2.5 array). Droplet digital PCR (Bio‐Rad QX200 Droplet Digital PCR system) was used to estimate the sensitivity of our scDNA‐seq platform.Results:Median 6,786 cells/sample were sequenced with median allele drop‐out rate of 7.2% (population frequency inferred from commonly heterozygous SNP loci). Each amplicon was covered at a median 26x/cell. The scDNA‐seq detected all of the bulk NGS‐confirmed mutations. RUNX1 and FLT3 mutations were frequently detected as homozygous mutations and concurrent SNP array analysis detected copy number neutral loss‐of‐heterozygosity of the mutant loci, which likely resulted in homozygous calls. The scDNA‐seq also uniquely detected driver mutations that were not detected by the bulk NGS but were confirmed by droplet digital PCR, suggesting that scDNA‐seq is more sensitive than the conventional bulk NGS.scDNA‐seq data unambiguously visualized the single‐cell level co‐occurrence of driver mutations (i.e. DNMT3A/FLT3‐ITD/NPM1 and SRSF2/IDH2), confirming the cooperative function of these mutations. scDNA‐seq data also revealed the cellular‐level mutual exclusivity between functionally redundant mutations such as IDH1/IDH2, FLT3‐ITD/TKD and NRAS/KRAS (Figure 1).Inference of phylogenetic trees using SCITE algorithm (Jahn, et al. Genome Biology 2016) uncovered distinct patterns of clonal evolution in AML. The majority of the cases showed a linear evolution pattern where the founder mutations linearly acquired sub‐clonal mutations in a step‐wise manner. We also detected convergent evolution in some cases where functionally similar driver mutations were acquired in parallel. DNMT3A, IDH1, IDH2 and U2AF1 mutations were frequently detected as trunk mutations, whereas FLT3, NRAS, and NPM1 mutations were usually detected as branch mutations. Analysis of longitudinal samples from 15 patients revealed the remodeling of clonal architecture in AML. For example, scDNA‐seq data for a previously‐untreated therapy‐related AML case revealed that a FLT3p.D835Y clone, which was originally presented as a small sub‐clone, survived the induction therapy consisting of azacitidine and sorafenib, and significantly expanded at relapse, while all other FLT3 and KRAS mutations were eradicated. This clonal remodeling is consistent with differential sensitivity of various FLT3 mutations to sorafenib (Smith et al. Leukemia 2015), of which FLT3 p.D835Y mutation has been shown to be more resistant to sorafenib compared to p.D835E and ITD mutations, demonstrating the differential behavior of sub‐clones and clonal selection under molecularly targeted therapy (Figure 2).Summary/Conclusion:We performed a high‐throughput scDNA‐seq and described a comprehensive landscape of driver mutations and detailed clonal evolution history in AML at the single‐cell resolution.image

Amplicon Based Panel Targeted Resequencing Identified ZRSR2 As a Potential New Favorable Marker in Pediatric AML

Introduction:Acute myeloid leukemia (AML) is one of the most threatening malignancies in children and adolescents. The accumulation of mutations in leukemia stem cells (LSC) is believed to lead to the development of leukemia. Cyto- and molecular genetics already identified several aberrations which are relevant in leukemogenesis, prognosis and therapy. Nevertheless, the molecular landscape and clonal evolution of AML and its clinical relevance, especially for pediatric patients, is not yet well described. Next Generation Sequencing (NGS) as an emerging sequencing technology provides the possibility to generate sequence data of high quality and detect genetic aberrations in a minimum of time. The aim of this study was to apply amplicon based panel targeted resequencing by using the TruSight Myeloid Panel (Illumina) to identify variants in 54 genes.MethodsPatients:In total 150 samples derived from pediatric patients diagnosed with AML at the time of initial diagnosis or relapse were analysed regarding their mutational profile. All patients treated according to the AML-BFM therapy protocols (n=103) were chosen to determine the potential impact in prognosis.Sequencing and analysis of variantsSequencing with the TruSight Myeloid panel (Illumina) was performed on a MiseqDX. The sequencing panel is designed to identify somatic mutations associated with myeloid malignancies in 54 genes. To define variants, we excluded intronic, synonymous and variants with an allele frequency below 5% and a read depth below 50 reads. False positive variants were excluded by including healthy donors and reference samples.Variants were detected and analysed using Variant studio (Illumina) and Sophia DDM (Sophia Genetics). Almost all variants were detectable in both software, although great insertions and deletions were detectable only by Sophia DDM.ResultsIn the cohort of 150 patients, we detected 408 mutations in the 54 genes included in the panel (fig. 1). 26% of the patients showed more than 4, 24% 3, 24% 2, 17% 1 and 9% of the patients showed 0 mutations. Four and more mutations occurred mostly in AML FAB M1 (n=17) and patients with a complex karyotype (n=6). Treatment related AML show less mutations compared to primary AML.Within the group of patients treated according the 1st line AML-BFM protocol (n=103), CEBPA, FLT3, KIT, NRAS, KRAS, NPM1 or WT1 mutations did not have prognostic relevance. Interestingly, we were able to detect mutations in ZRSR2 in 21 patients in total (SNVs in 6, InDels in 9 and splice acceptor variants in 6 patients). 15 patients were part of the group of patients who were treated according to the 1st line AML-BFM protocol. ZRSR2 encodes an essential splicing factor and the encoded protein associates with the U2 auxiliary factor heterodimer and may play a role in network interactions during spliceosome assembly [RefSeq 2008]. The presence of a ZRSR2 mutation seems to be associated with better EFS and lower cumulative incidence of relapse, respectively (fig.2). Even if patients with favourable cytogenetics were excluded, patients with mutated ZRSR2 might have better EFS (fig.2).Conclusions:Amplicon based panel targeted resequencing with the TruSight Myeloid panel provides the possibility to detect mutations in 54 genes associated to myeloid malignancies within 3 days. This will enable a faster and possible more precise characterisation of pediatric AML, either for risk group stratification or the addition of more specific treatment options. Due to the limited number of patients, the results concerning the prognostic relevance of ZRSR2 need to be confirmed in a larger patient group. [Display omitted] [Display omitted] [Display omitted] DisclosuresReinhardt:Jazz Pharma: Other: Travel Accomodation; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

DNMT3A Mutations in AML Patients: Prognostic Impact and Comparative Analysis of Mutations Burden in Diagnostic Samples, after Standard Therapy, and after Allogeneic Stem Cell Transplantation

DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A) mutations is one of the most frequent somatic mutations in acute myeloid leukemia (AML). Although DNMT3A mutations have been studied extensively, influence of mutations on AML prognosis and their role on the pathogenesis of AML remains not completely clear. Little is known about the significance of mutations burden in the time of diagnosis on the clinical and prognostic features of AML. Recently discovered persistence of DNMT3A mutations in complete remission after standard therapy indicates the presence of mutation in early pre-leukemic stem cells. In this aspect, it would be essential to analyze the existence of DNMT3A-positive cells after allogeneic stem cell transplantation (alloSCT).Using quantitative PCR, we analyzed mutations burden in diagnostic samples obtained from 65 AML patients with DNMT3A mutation. Then follow up samples were analyzed repeatedly, after induction, consolidation therapy, and after alloSCT with a mean follow-up of 33 months. Using well-established markers of minimal residual disease and donor engraftment, we monitored DNMT3A stability during complete molecular remission (CMR). In addition, we analyzed the combination of DNMT3A mutation with others mutations, such as FLT3, NPM1, IDH1, IDH,and others, at the time of diagnosis, as well as in relapse of AML. Finally, we analyzed the prognostic impact of DNMT3A mutations depending on the distinct combination of mutations with other genetic markers. For this, 150 matched-pairs AML patients without DNMT3A mutation were analyzed.We have found considerable variation of mutations burden (from 1% till 90%) in diagnostic samples. Although, the prognostic impact of mutations burden in diagnostic samples have not been verify, we have found a significant associations of higher mutations burden with M4/M5 FAB variant of AML (p=0,04) and presence of NPM1 mutation (p=0,023).Persistence of DNMT3A mutations in CMR after standard chemotherapy was found in all patients. The loss of correlation between DNMT3A and others could be explained by sub-clone architecture and clonal evolution of AML. After alloSCT, DNMT3A mutations were not discovered in patients with CMR and complete donor chimerism. This data suggests the removal of leukemic stem cells after alloSCT and indicate the importance of alloSCT for high risk AML patients. In relapse of leukemia, all samples showed an increasing of DNMT3A mutated alleles.In addition, matched-pairs analysis suggests that DNMT3A, particularly when accompanied by FLT3-ITD, is a significant prognostic factor for inferior outcome, even after alloSCT.We conclude that long-term quantitative monitoring of DNMT3A mutations could provide additional prognostic information and could explain the role of mutations in development and progression of AML. DisclosuresPezzutto:Cellgene, Novartis, Roche, Gilead: Membership on an entity's Board of Directors or advisory committees.

The Pro-Inflammatory Cytokine Interleukin-1 Is Essential for Clonal Expansion and Disease Progression in Acute Myeloid Leukemia

The Pro-Inflammatory Cytokine Interleukin-1 Is Essential for Clonal Expansion and Disease Progression in Acute Myeloid Leukemia

Next Generation Sequencing of Acute Myeloid Leukemia: Influencing Prognosis

Acute myeloid leukemia (AML) is a clonal disorder of the blood forming cells characterized by accumulation of immature blast cells in the bone marrow and peripheral blood. Being a heterogeneous disease, AML has been the subject of numerous studies that focus on unraveling the clinical, cellular and molecular variations with the aim to better understand and treat the disease. Cytogenetic-risk stratification of AML is well established and commonly used by clinicians in therapeutic management of cases with chromosomal abnormalities. Successive inclusion of novel molecular abnormalities has substantially modified the classification and understanding of AML in the past decade. With the advent of next generation sequencing (NGS) technologies the discovery of novel molecular abnormalities has accelerated. NGS has been successfully used in several studies and has provided an unprecedented overview of molecular aberrations as well as the underlying clonal evolution in AML. The extended spectrum of abnormalities discovered by NGS is currently under extensive validation for their prognostic and therapeutic values. In this review we highlight the recent advances in the understanding of AML in the NGS era.

Preleukemic mutations in human acute myeloid leukemia affect epigenetic regulators and persist in remission

Cancer is widely characterized by the sequential acquisition of genetic lesions in a single lineage of cells. Our previous studies have shown that, in acute myeloid leukemia (AML), mutation acquisition occurs in functionally normal hematopoietic stem cells (HSCs). These preleukemic HSCs harbor some, but not all, of the mutations found in the leukemic cells. We report here the identification of patterns of mutation acquisition in human AML. Our findings support a model in which mutations in "landscaping" genes, involved in global chromatin changes such as DNA methylation, histone modification, and chromatin looping, occur early in the evolution of AML, whereas mutations in "proliferative" genes occur late. Additionally, we analyze the persistence of preleukemic mutations in patients in remission and find CD34+ progenitor cells and various mature cells that harbor preleukemic mutations. These findings indicate that preleukemic HSCs can survive induction chemotherapy, identifying these cells as a reservoir for the reevolution of relapsed disease. Finally, through the study of several cases of relapsed AML, we demonstrate various evolutionary patterns for the generation of relapsed disease and show that some of these patterns are consistent with involvement of preleukemic HSCs. These findings provide key insights into the monitoring of minimal residual disease and the identification of therapeutic targets in human AML.

Genetic Profiling By Targeted, Deep Resequencing Confirms That a Murine Xenograft Model Of Acute Myeloid Leukemia (AML) Recapitulates The Mutational Landscape Of The Human Disease and Provides Evidence For Clonal Heterogeneity and Clonal Evolution

BackgroundA growing number of recurrent gene mutations have been identified in AML through novel sequencing techniques. To uncover the functional consequences of these mutations and identify novel therapeutic targets, adequate model systems are needed. Such models should recapitulate the mutational landscape and genetic heterogeneity of AML as closely as possible. In this respect, analyses of primary tumor cells are superior to established permanent cell lines. Xenografts of primary AML blasts in immunodeficient mice thus may represent a valuable platform for functional analyses on a wide spectrum of AML subtypes. However, it has not been determined whether the mutational architecture of AML xenografts faithfully represents the human disease they originate from. MethodsFresh bone marrow or peripheral blood samples were obtained from adult AML patients. One to ten million cells were injected intravenously or intrafemorally into recipient NOD-scid IL2R-gammanull(NSG) mice. Engraftment was monitored by serial immunophenotyping using anti-human CD33 and anti-human CD45 antibodies. Mice showing >35% human cells in the peripheral blood or signs of disease were sacrificed, and xenotransplanted cells re-isolated from femurs and spleens. Using genomic DNA from xenografts and paired primary human specimens, we profiled mutations in 44 genes recurrently mutated in human hematologic malignancies by targeted, deep amplicon resequencing (Agilent Haloplex / Illumina MiSeq). ResultsTo date, 4 pairs of primary AML specimens and matched xenografts have been genetically characterized by targeted resequencing. We obtained between 480k and 1100k paired-end reads (2x250bp) per sample, resulting in >30x coverage for >98.7% of the target sequence. The median coverage for individual target regions ranged from 116-fold (CEBPA) to ∼5200-fold (FLT3).In each patient specimen, 2 to 5 known AML-associated driver mutations were identified in our panel of 44 genes. The Figure shows variant allele frequencies (VAFs) for mutations and known germline polymorphisms (SNPs; dbSNP database v137), in the patient samples and matched xenografts. All mutations that were present in the primary patient samples with a VAF of >10% were also found in the matched xenograft. None of the xenografts acquired new mutations that were undetectable in the original patient specimen. However, each sample pair showed evidence for clonal diversity and clonal evolution. In patient AML361, NPM1, DNMT3A and BCOR mutations were detected at a VAF slightly below .5, consistent with heterozygous mutations present in most cells in the primary specimen. Additionally, a FLT3-internal tandem duplication (ITD) was present at a lower VAF, likely representing a subclonal mutation. In the corresponding xenograft, the FLT3-ITD was observed in a significantly larger fraction of cells, suggesting that the FLT3-mutated clone had a relative growth advantage. Similarly, patient AML393 carried a subclone with mutated BCOR that became the dominant clone in the xenograft. Conversely, in patients AML372 and AML373 we found subclonal NRAS mutations (VAF, 6% and 7%, respectively) that were undetectable in the matched xenografts, indicating that the NRAS-mutated subclones did not engraft. We are currently studying parallel lines of xenografts generated from the same patient, followed by serial re-passaging in NSG mice, to better characterize growth patterns of such patient-derived subclones in our model. Updated results will be presented at the meeting. [Display omitted] Genotypes for known germline SNPs were fully concordant between the human specimens and matched xenografts in 3/4 pairs. The fourth patient (AML393), who had AML relapse after an allogeneic stem cell transplant, showed several SNPs with a low VAF that were not identified in the corresponding xenograft. These SNPs most likely originate from residual donor hematopoiesis in the patient, indicating that non-malignant cells of donor origin did not engraft in the NSG mouse. ConclusionXenografts of primary AML blasts in NSG mice recapitulate the patterns of gene mutations observed in AML patients, and thus provide an opportunity to study the biology of various genetic AML subgroups. Deep, targeted amplicon resequencing can sensitively detect subclonal driver mutations, and in conjunction with the xenograft model can be used to study clonal diversity and clonal evolution in AML. Disclosures:Greif:Illumina:Honoraria.

The Origin and Evolution of Mutations in Acute Myeloid Leukemia

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.

Novel Chromosomal Aberration as Evidence of Clonal Evolution in a Case of Relapsed Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of diseases with a multitude of molecular genetic aberrations and variable clinical outcome. Clonal chromosomal abnormalities have been identified in over 50% of AML cases, and have been regarded as one of the most important prognostic markers. We present a case of a 28-year-old Caucasian woman with AML without maturation, diploid karyotype, that was resistant to multiple chemotherapies and relapsed after matched unrelated stem cell transplantation. Conventional cytogenetic analysis performed on bone marrow specimens revealed 46,XX,t(2;16)(p21;p11.2),t(11;14)(p13;p11.2). The t(11;14)(p13;p11.2) was confirmed by fluorescence in situ hybridization using a whole chromosome paint probe for chromosome 11. Morphologically, the bone marrow was hypercellular with trilineage hypoplasia and 84% blasts. Flow cytometry analysis showed that the blasts were of myeloid immunophenotype. Molecular studies detected internal tandem duplication of the FLT3 gene and a mutation in exon 12 of the NPM1 gene. The patient then received monotherapy with AC220, achieved a brief remission, and died of relapsed disease 23 months after initial diagnosis. This is the first report of this novel clonal chromosome aberration as evidence of clonal evolution in AML. [N A J Med Sci. 2012;5(1):48-50.]