AML with complex karyotype: extreme genomic complexity revealed by combined long-read sequencing and Hi-C technology.

Acute myeloid leukemia with complex karyotype (CK-AML) is associated with poor prognosis, which is only in part explained by underlying TP53 mutations. Especially in the presence of complex chromosomal rearrangements, such as chromothripsis, the outcome of CK-AML is dismal. However, this degree of complexity of genomic rearrangements contributes to the leukemogenic phenotype and treatment resistance of CK-AML remains largely unknown. Applying an integrative workflow for the detection of structural variants (SVs) based on Oxford Nanopore (ONT) genomic DNA long-read sequencing (gDNA-LRS) and high-throughput chromosome confirmation capture (Hi-C) in a well-defined cohort of CK-AML identified regions with an extreme density of SVs. These rearrangements consisted to a large degree of focal amplifications enriched in the proximity of mammalian-wide interspersed repeat elements, which often result in oncogenic fusion transcripts, such as USP7::MVD, or the deregulation of oncogenic driver genes as confirmed by RNA-seq and ONT direct complementary DNA sequencing. We termed this novel phenomenon chromocataclysm. Thus, our integrative SV detection workflow combing gDNA-LRS and Hi-C enables to unravel complex genomic rearrangements at a very high resolution in regions hard to analyze by conventional sequencing technology, thereby providing an important tool to identify novel important drivers underlying cancer with complex karyotypic changes.

Molecular landscape and prognostic impact of FLT3-ITD insertion site in acute myeloid leukemia: RATIFY study results.

In acute myeloid leukemia (AML) internal tandem duplications of the FLT3 gene (FLT3-ITD) are associated with poor prognosis. Retrospectively, we investigated the prognostic and predictive impact of FLT3-ITD insertion site (IS) in 452 patients randomized within the RATIFY trial, which evaluated midostaurin additionally to intensive chemotherapy. Next-generation sequencing identified 908 ITDs, with 643 IS in the juxtamembrane domain (JMD) and 265 IS in the tyrosine kinase domain-1 (TKD1). According to IS, patients were categorized as JMDsole (n = 251, 55%), JMD and TKD1 (JMD/TKD1; n = 117, 26%), and TKD1sole (n = 84, 19%). While clinical variables did not differ among the 3 groups, NPM1 mutation was correlated with JMDsole (P = 0.028). Overall survival (OS) differed significantly, with estimated 4-year OS probabilities of 0.44, 0.50, and 0.30 for JMDsole, JMD/TKD1, and TKD1sole, respectively (P = 0.032). Multivariate (cause-specific) Cox models for OS and cumulative incidence of relapse using allogeneic hematopoietic cell transplantation (HCT) in first complete remission as a time-dependent variable identified TKD1sole as unfavorable and HCT as favorable factors. In addition, Midostaurin exerted a significant benefit only for JMDsole. Our results confirm the distinct molecular heterogeneity of FLT3-ITD and the negative prognostic impact of TKD1 IS in AML that was not overcome by midostaurin.

The retinoic acid receptor co-factor NRIP1 is uniquely upregulated and represents a therapeutic target in acute myeloid leukemia with chromosome 3q rearrangements.

Aberrant expression of Ecotropic Viral Integration Site 1 (EVI1) is a hallmark of acute myeloid leukemia (AML) with inv(3) or t(3;3), which is a disease subtype with especially poor outcome. In studying transcriptomes from AML patients with chromosome 3q rearrangements, we identified a significant upregulation of the Nuclear Receptor Interacting Protein 1 (NRIP1) as well as its adjacent non-coding RNA LOC101927745. Utilizing transcriptomic and epigenomic data from over 900 primary samples from patients as well as genetic and transcriptional engineering approaches, we have identified several mechanisms that can lead to upregulation of NRIP1 in AML. We hypothesize that the LOC101927745 transcription start site harbors a context-dependent enhancer that is bound by EVI1, causing upregulation of NRIP1 in AML with chromosome 3 abnormalities. Furthermore, we showed that NRIP1 knockdown negatively affects the proliferation and survival of 3qrearranged AML cells and increases their sensitivity to all-trans retinoic acid, suggesting that NRIP1 is relevant for the pathogenesis of inv(3)/t(3;3) AML and could serve as a novel therapeutic target in myeloid malignancies with 3q abnormalities.

Clonal evolution of acute myeloid leukemia with FLT3-ITD mutation under treatment with midostaurin.

In the international randomized phase 3 RATIFY (Randomized AML Trial In FLT3 in patients less than 60 Years old) trial, the multikinase inhibitor midostaurin significantly improved overall and event-free survival in patients 18 to 59 years of age with FLT3-mutated acute myeloid leukemia (AML). However, only 59% of patients in the midostaurin arm achieved protocol-specified complete remission (CR), and almost half of patients achieving CR relapsed. To explore underlying mechanisms of resistance, we studied patterns of clonal evolution in patients with FLT3-internal tandem duplications (ITD)-positive AML who were entered in the RATIFY or German-Austrian Acute Myeloid Leukemia Study Group 16-10 trial and received treatment with midostaurin. To this end, paired samples from 54 patients obtained at time of diagnosis and at time of either relapsed or refractory disease were analyzed using conventional Genescan-based testing for FLT3-ITD and whole exome sequencing. At the time of disease resistance or progression, almost half of the patients (46%) became FLT3-ITD negative but acquired mutations in signaling pathways (eg, MAPK), thereby providing a new proliferative advantage. In cases with FLT3-ITD persistence, the selection of resistant ITD clones was found in 11% as potential drivers of disease. In 32% of cases, no FLT3-ITD mutational change was observed, suggesting either resistance mechanisms bypassing FLT3 inhibition or loss of midostaurin inhibitory activity because of inadequate drug levels. In summary, our study provides novel insights into the clonal evolution and resistance mechanisms of FLT3-ITD-mutated AML under treatment with midostaurin in combination with intensive chemotherapy.

Genomic heterogeneity in core-binding factor acute myeloid leukemia and its clinical implication.

Core-binding factor (CBF) acute myeloid leukemia (AML) encompasses AML with inv(16)(p13.1q22) and AML with t(8;21)(q22;q22.1). Despite sharing a common pathogenic mechanism involving rearrangements of the CBF transcriptional complex, there is growing evidence for considerable genotypic heterogeneity. We comprehensively characterized the mutational landscape of 350 adult CBF-AML [inv(16): n = 160, t(8;21): n = 190] performing targeted sequencing of 230 myeloid cancer-associated genes. Apart from common mutations in signaling genes, mainly NRAS, KIT, and FLT3, both CBF-AML entities demonstrated a remarkably diverse pattern with respect to the underlying cooperating molecular events, in particular in genes encoding for epigenetic modifiers and the cohesin complex. In addition, recurrent mutations in novel collaborating candidate genes such as SRCAP (5% overall) and DNM2 (6% of t(8;21) AML) were identified. Moreover, aberrations altering transcription and differentiation occurred at earlier leukemic stages and preceded mutations impairing proliferation. Lasso-penalized models revealed an inferior prognosis for t(8;21) AML, trisomy 8, as well as FLT3 and KIT exon 17 mutations, whereas NRAS and WT1 mutations conferred superior prognosis. Interestingly, clonal heterogeneity was associated with a favorable prognosis. When entering mutations by functional groups in the model, mutations in genes of the methylation group (ie, DNMT3A, TET2) had a strong negative prognostic impact.

Impact of gemtuzumab ozogamicin on MRD and relapse risk in patients with NPM1-mutated AML: results from the AMLSG 09-09 trial.

Monitoring of measurable residual disease (MRD) provides prognostic information in patients with Nucleophosmin1-mutated (NPM1mut) acute myeloid leukemia (AML) and represents a powerful tool to evaluate treatment effects within clinical trials. We determined NPM1mut transcript levels (TLs) by quantitative reverse-transcription polymerase chain reaction and evaluated the prognostic impact of NPM1mut MRD and the effect of gemtuzumab ozogamicin (GO) on NPM1mut TLs and the cumulative incidence of relapse (CIR) in patients with NPM1mut AML enrolled in the randomized phase 3 AMLSG 09-09 trial. A total of 3733 bone marrow (BM) samples and 3793 peripheral blood (PB) samples from 469 patients were analyzed. NPM1mut TL log10 reduction ≥ 3 and achievement of MRD negativity in BM and PB were significantly associated with a lower CIR rate, after 2 treatment cycles and at end of treatment (EOT). In multivariate analyses, MRD positivity was consistently revealed to be a poor prognostic factor in BM and PB. With regard to treatment effect, the median NPM1mut TLs were significantly lower in the GO-Arm across all treatment cycles, resulting in a significantly greater proportion of patients achieving MRD negativity at EOT (56% vs 41%; P = .01). The better reduction in NPM1mut TLs after 2 treatment cycles in MRD positive patients by the addition of GO led to a significantly lower CIR rate (4-year CIR, 29.3% vs 45.7%, P = .009). In conclusion, the addition of GO to intensive chemotherapy in NPM1mut AML resulted in a significantly better reduction in NPM1mut TLs across all treatment cycles, leading to a significantly lower relapse rate.

Rescue of Non-Informative Circulating Tumor DNA to Monitor the Mutational Landscape in NSCLC.

In non-small cell lung cancer (NSCLC) the usage of plasma-derived circulating tumor DNA (ctDNA) have come into focus to obtain a comprehensive genetic profile of a given lung cancer. Despite the usage of specific sampling tubes, archived plasma samples as well as inappropriately treated blood samples still cause a loss of information due to cell lysis and contamination with cellular DNA. Our aim was to establish a reliable protocol to rescue ctDNA from such non-informative samples to monitor the mutational landscape in NSCLC. As a proof-of-concept study we used archived plasma samples derived from whole blood EDTA samples of 51 patients suffering from NSCLC. Analysis of the isolated plasma DNA determined only a small fraction of ctDNA in a range of 90-250 bp. By applying a specific purification procedure, we were able to increase the informative ctDNA content and improve in a cohort of 42 patients the detection of driver mutations from 32% to 79% of the mutations found in tissue biopsies. Thus, we present here an easy to perform, time and cost effective procedure to rescue non-informative ctDNA samples, which is sufficient to detect oncogenic mutations in NGS approaches and is therefore a valuable technical improvement for laboratories handling liquid biopsy samples.

Functional Classification of TP53 Mutations in Acute Myeloid Leukemia.

Mutations of the TP53 gene occur in a subset of patients with acute myeloid leukemia (AML) and confer an exceedingly adverse prognosis. However, whether different types of TP53 mutations exert a uniformly poor outcome has not been investigated yet. Here, we addressed this issue by analyzing data of 1537 patients intensively treated within protocols of the German-Austrian AML study group. We classified TP53 mutations depending on their impact on protein structure and according to the evolutionary action (EAp53) score and the relative fitness score (RFS). In 98/1537 (6.4%) patients, 108 TP53 mutations were detected. While the discrimination depending on the protein structure and the EAp53 score did not show a survival difference, patients with low-risk and high-risk AML-specific RFS showed a different overall survival (OS; median, 12.9 versus 5.5 months, p = 0.017) and event-free survival (EFS; median, 7.3 versus 5.2 months, p = 0.054). In multivariable analyses adjusting for age, gender, white blood cell count, cytogenetic risk, type of AML, and TP53 variant allele frequency, these differences were statistically significant for both OS (HR, 2.14; 95% CI, 1.15-4.0; p = 0.017) and EFS (HR, 1.97; 95% CI, 1.06-3.69; p = 0.033). We conclude that the AML-specific RFS is of prognostic value in patients with TP53-mutated AML and a useful tool for therapeutic decision-making.

Therapeutic targeting of preleukemia cells in a mouse model of NPM1 mutant acute myeloid leukemia.

The initiating mutations that contribute to cancer development are sometimes present in premalignant cells. Whether therapies targeting these mutations can eradicate premalignant cells is unclear. Acute myeloid leukemia (AML) is an attractive system for investigating the effect of preventative treatment because this disease is often preceded by a premalignant state (clonal hematopoiesis or myelodysplastic syndrome). In Npm1c/Dnmt3a mutant knock-in mice, a model of AML development, leukemia is preceded by a period of extended myeloid progenitor cell proliferation and self-renewal. We found that this self-renewal can be reversed by oral administration of a small molecule (VTP-50469) that targets the MLL1-Menin chromatin complex. These preclinical results support the hypothesis that individuals at high risk of developing AML might benefit from targeted epigenetic therapy in a preventative setting.

Measurable residual disease monitoring in acute myeloid leukemia with t(8;21)(q22;q22.1): results from the AML Study Group.

We performed serial measurable residual disease (MRD) monitoring in bone marrow (BM) and peripheral blood (PB) samples of 155 intensively treated patients with RUNX1-RUNX1T1+ AML, using a qRT-PC-based assay with a sensitivity of up to 10-6. We assessed both reduction of RUNX1-RUNX1T1 transcript levels (TLs) and achievement of MRD negativity (MRD-) for impact on prognosis. Achievement of MR2.5 (>2.5 log reduction) after treatment cycle 1 and achievement of MR3.0 after treatment cycle 2 were significantly associated with a reduced risk of relapse (P = .034 and P = .028, respectively). After completion of therapy, achievement of MRD- in both BM and PB was an independent, favorable prognostic factor in cumulative incidence of relapse (4-year cumulative incidence relapse: BM, 17% vs 36%, P = .021; PB, 23% vs 55%, P = .001) and overall survival (4-year overall survival rate BM, 93% vs 70%, P = .007; PB, 87% vs 47%, P < .0001). Finally, during follow-up, serial qRT-PCR analyses allowed prediction of relapse in 77% of patients exceeding a cutoff value of 150 RUNX1-RUNX1T1 TLs in BM, and in 84% of patients exceeding a value of 50 RUNX1-RUNX1T1 TLs in PB. The KIT mutation was a significant factor predicting a lower CR rate and inferior outcome, but its prognostic impact was outweighed by RUNX1-RUNX1T1 TLs during treatment. Virtually all relapses occurred within 1 year after the end of treatment, with a very short latency from molecular to morphologic relapse, necessitating MRD assessment at short intervals during this time period. Based on our data, we propose a refined practical guideline for MRD assessment in RUNX1-RUNX1T1+ AML.

A dominant-negative effect drives selection of TP53 missense mutations in myeloid malignancies.

TP53, which encodes the tumor suppressor p53, is the most frequently mutated gene in human cancer. The selective pressures shaping its mutational spectrum, dominated by missense mutations, are enigmatic, and neomorphic gain-of-function (GOF) activities have been implicated. We used CRISPR-Cas9 to generate isogenic human leukemia cell lines of the most common TP53 missense mutations. Functional, DNA-binding, and transcriptional analyses revealed loss of function but no GOF effects. Comprehensive mutational scanning of p53 single-amino acid variants demonstrated that missense variants in the DNA-binding domain exert a dominant-negative effect (DNE). In mice, the DNE of p53 missense variants confers a selective advantage to hematopoietic cells on DNA damage. Analysis of clinical outcomes in patients with acute myeloid leukemia showed no evidence of GOF for TP53 missense mutations. Thus, a DNE is the primary unit of selection for TP53 missense mutations in myeloid malignancies.

Clonal evolution patterns in acute myeloid leukemia with NPM1 mutation.

Mutations in the nucleophosmin 1 (NPM1) gene are considered founder mutations in the pathogenesis of acute myeloid leukemia (AML). To characterize the genetic composition of NPM1 mutated (NPM1mut) AML, we assess mutation status of five recurrently mutated oncogenes in 129 paired NPM1mut samples obtained at diagnosis and relapse. We find a substantial shift in the genetic pattern from diagnosis to relapse including NPM1mut loss (n = 11). To better understand these NPM1mut loss cases, we perform whole exome sequencing (WES) and RNA-Seq. At the time of relapse, NPM1mut loss patients (pts) feature distinct mutational patterns that share almost no somatic mutation with the corresponding diagnosis sample and impact different signaling pathways. In contrast, profiles of pts with persistent NPM1mut are reflected by a high overlap of mutations between diagnosis and relapse. Our findings confirm that relapse often originates from persistent leukemic clones, though NPM1mut loss cases suggest a second "de novo" or treatment-associated AML (tAML) as alternative cause of relapse.

New Targeted Agents in Acute Myeloid Leukemia: New Hope on the Rise.

The therapeutic approach for acute myeloid leukemia (AML) remains challenging, since over the last four decades a stagnation in standard cytotoxic treatment has been observed. But within recent years, remarkable advances in the understanding of the molecular heterogeneity and complexity of this disease have led to the identification of novel therapeutic targets. In the last two years, seven new targeted agents (midostaurin, gilteritinib, enasidenib, ivosidenib, glasdegib, venetoclax and gemtuzumab ozogamicin) have received US Food and Drug Administration (FDA) approval for the treatment of AML. These drugs did not just prove to have a clinical benefit as single agents but have especially improved AML patient outcomes if they are combined with conventional therapy. In this review, we will focus on currently approved and promising upcoming agents and we will discuss controversial aspects and limitations of targeted treatment strategies.

Analysis of the CDK4/6 Cell Cycle Pathway in Leiomyosarcomas as a Potential Target for Inhibition by Palbociclib.

Leiomyosarcoma (LMS) is characterized by high genomic complexity, and to date, no specific targeted therapy is available. In a genome-wide approach, we profiled genomic aberrations in a small cohort of eight primary tumours, two relapses, and eight metastases across nine different patients. We identified CDK4 amplification as a recurrent alteration in 5 out of 18 samples (27.8%). It has been previously shown that the LMS cell line SK-LMS-1 has a defect in the p16 pathway and that this cell line can be inhibited by the CDK4 and CDK6 inhibitor palbociclib. For SK-LMS-1 we confirm and for SK-UT-1 we show that both LMS cell lines express CDK4 and that, in addition, strong CDK6 expression is seen in SK-LMS-1, whereas Rb was expressed in SK-LMS-1 but not in SK-UT-1. We confirm that inhibition of SK-LMS-1 with palbociclib led to a strong decrease in protein levels of Phospho-Rb (Ser780), a decreased cell proliferation, and G0/G1-phase arrest with decreased S/G2 fractions. SK-UT-1 did not respond to palbociclib inhibition. To compare these in vitro findings with patient tissue samples, a p16, CDK4, CDK6, and p-Rb immunohistochemical staining assay of a large LMS cohort (n=99 patients with 159 samples) was performed assigning a potential responder phenotype to each patient, which we identified in 29 out of 99 (29.3%) patients. Taken together, these data show that CDK4/6 inhibitors may offer a new option for targeted therapy in a subset of LMS patients.

Clinical implications of subclonal TP53 mutations in acute myeloid leukemia.

The role of subclonal TP53 mutations, defined by a variant allele frequency of <20%, has not been addressed in acute myeloid leukemia yet. We, therefore, analyzed their prognostic value in a cohort of 1,537 patients with newly diagnosed disease, prospectively treated within three trials of the "German-Austrian Acute Myeloid Leukemia Study Group". Mutational analysis was performed by targeted deep sequencing and patients with TP53 mutations were categorized by their variant allele frequency into groups with frequencies >40%, 20%-40% and <20%. A total of 108 TP53 mutations were found in 98 patients (6.4%). Among these, 61 patients had variant allele frequencies >40%, 19 had variant allele frequencies between 20%-40% and 18 had frequencies <20%. Compared to specimens with clonal TP53 mutations, those with subclonal ones showed significantly fewer complex karyotypes and chromosomal losses. In either TP53-mutated group, patients experienced significantly fewer complete responses (P<0.001) and had worse overall and event-free survival rates (P<0.0001). In Cox regression analyses adjusting for age, white blood cell count, cytogenetic risk and type of acute myeloid leukemia, the adverse prognostic effect of TP53 mutations remained significant for all TP53-mutated subgroups. These data suggest that subclonal TP53 mutations are a novel prognostic parameter in acute myeloid leukemia and emphasize the usefulness of next-generation sequencing technologies for risk stratification in this disorder. The study was registered at ClinicalTrials.gov with number NCT00146120.

Epigenetic therapy: azacytidine and decitabine in acute myeloid leukemia.

INTRODUCTION: The majority of patients with acute myeloid leukemia (AML) are older and exhibit a poor prognosis even after intensive therapy. Inducing differentiation and apoptosis of leukemic blasts by DNA-hypomethylating agents, like e.g. azacytidine (AZA) and decitabine (DAC), represent well-tolerated alternative treatment approaches. Both agents show convincing response as single agents in AML. However, there is a lack of knowledge regarding molecular mechanisms and predictive biomarkers for these agents. Areas covered: This review will (i) provide an overview of the current knowledge of molecular mechanisms underlying the action of these drugs, (ii) report promising predictive biomarkers, (iii) elude on new combined treatment options, and (iv) discuss novel approaches to improve outcomes. A literature search was performed using PubMed to find recent major publications, which provide biological and clinical research about epigenetic therapy in AML patients. Expert commentary: Numerous studies have demonstrated that HMA therapy with AZA or DAC may lead to significant response rates, even in pre-treated patients. Nevertheless, there is still an unmet need to further improve outcome in elderly AML patients. Therefore, novel treatment combinations are needed and some of them, such as AZA plus venetoclax, already show promising results.

Protein phosphatase 4 regulatory subunit 2 (PPP4R2) is recurrently deleted in acute myeloid leukemia and required for efficient DNA double strand break repair.

We have previously identified a recurrent deletion at chromosomal band 3p14.1-p13 in patients with acute myeloid leukemia (AML). Among eight protein-coding genes, this microdeletion affects the protein phosphatase 4 regulatory subunit 2 (PPP4R2), which plays an important role in DNA damage response (DDR). Investigation of mRNA expression during murine myelopoiesis determined that Ppp4r2 is higher expressed in more primitive hematopoietic cells. PPP4R2 expression in primary AML samples compared to healthy bone marrow was significantly lower, particularly in patients with 3p microdeletion or complex karyotype. To identify a functional role of PPP4R2 in hematopoiesis and leukemia, we genetically inactivated Ppp4r2 by RNAi in murine hematopoietic stem and progenitor cells and murine myeloid leukemia. Furthermore, we ectopically expressed PPP4R2 in a deficient human myeloid leukemic cell line. While PPP4R2 is involved in DDR of both hematopoietic and leukemic cells, our findings indicate that PPP4R2 deficiency impairs de-phosphorylation of phosphorylated key DDR proteins KRAB-domain associated protein 1 (pKAP1), histone variant H2AX (γH2AX), tumor protein P53 (pP53), and replication protein A2 (pRPA2). Potential impact of affected DNA repair processes in primary AML cases with regard to differential PPP4R2 expression or 3p microdeletion is also supported by our results obtained by gene expression profiling and whole exome sequencing. Impaired DDR and increased DNA damage by PPP4R2 suppression is one possible mechanism by which the 3p microdeletion may contribute to the pathogenesis of AML. Further studies are warranted to determine the potential benefit of inefficient DNA repair upon PPP4R2 deletion to the development of therapeutic agents.