Nucleophosmin Mutations Research Articles

NPM1 and FLT3-ITD/TKD Gene Mutations in Acute Myeloid Leukemia.

Background: A number of mutations have been reported to occur in patients with acute myeloid leukemia (AML), of which NPM1 and FLT3 genes mutations are the commonest and have important diagnostic and therapeutic implications. Material and Methods: Molecular testing for NPM1 and FLT3 genes was performed in 92 de-novo AML patients. The frequency and characteristics of NPM1 and FLT3 mutations were analyzed. Results: Nucleophosmin 1(NPM1) and fms-like tyrosine kinase 3 (FLT3) mutations were seen in 22.8% and 16.3% of patients, respectively. Amongst FLT3 mutations, FLT3-ITD mutation was seen in 8.7% cases, FLT3-TKD in 5.4%, and FLT3-ITD+TKD in 2.2% cases. Certain associations between the gene mutations and clinical characteristics were found, including in NPM1 mutated group- female preponderance, higher incidence in M4/M5 categories and decreased expression of CD34 and HLA-DR; and in FLT3-ITD mutated group- higher age of presentation, higher total leucocyte count and blast percentage. Conclusion- AML patients with NPM1 and FLT3 mutations have differences in clinical and hematological features, which might represent their different molecular mechanism in leukemogenesis. The frequency of NPM1 and FLT3 mutations in this study was comparable to reports from Asian countries but lower than that reported from western countries. However, as the number of patients in the study was less, a larger number of patients need to be studied to corroborate these findings.

NPM1 mutant maintains ULK1 protein stability via TRAF6-dependent ubiquitination to promote autophagic cell survival in leukemia.

Nucleophosmin (NPM1) mutations are the most frequent genetic alteration in acute myeloid leukemia (AML) and aberrant cytoplasm-dislocated NPM1 mutant is a distinct biological characterization of this disease. Our group previously reported that NPM1 mutant elevated autophagy activity and autophagy activation contributed to leukemic cell survival. However, the molecular mechanisms by which cytoplasmic NPM1 mutant involving in the autophagy pathway has not been fully elucidated. Here, we showed that Unc-51-like kinase 1 (ULK1) as a core autophagy protein was highly expressed in NPM1-mA positive OCI-AML3 cells and primary NPM1-mutated AML blasts. Meanwhile, we found that NPM1-mA could interact with ULK1 protein and positively regulated ULK1 protein levels. Mechanically, NPM1-mA promoted TRAF6-dependent K63 ubiquitination and further maintained ULK1 stability and kinase activity via miR-146a. In addition, ULK1 high expression-mediated autophagy activation and facilitated to leukemic cell proliferation. Finally, we demonstrated that restoring ULK1 expression, ULK1 inhibitor SBI-0206965 treatment and using shULK1 partially rescued the effect of NPM1-mA on autophagy and cell survival. In conclusion, our findings suggest that NPM1 mutant interacts with ULK1, and thus, maintains its protein stability, which is required for NPM1 mutant-mediated autophagic cell survival. These data extend our understanding of the functions of NPM1 mutant in the regulation of autophagy activation in NPM1-mutated AML.

Preferential transcription of the mutated allele in NPM1 mutated acute myeloid leukaemia

Nucleophosmin is commonly both over-expressed and mutated in acute myeloid leukemia (AML). NPM1 mutations are always heterozygous. In addition, NPM1 has a number of different splice variants with the major variant encoded by exons 1–9 and 11–12 (NPM1.1). Further variants include NPM1.2 which lacks exons 8 and 10 and NPM1.3 which comprises exons 1–10 (and so lacks the region of sequence mutated in AML). In this study we quantified the expression of these three variants in 108 AML patient samples with and without NPM1 mutations and also assessed the level of expression from the wild-type and mutant alleles in variants NPM1.1 and NPM1.2. The results show that NPM1.1 is the most commonly expressed variant, however transcripts from wild-type and mutated alleles do not occur at equal levels, with a significant bias toward the mutated allele. Considering the involvement of mutant nucleophosmin in the progression and maintenance of AML, a bias towards mutated transcripts could have a significant impact on disease maintenance.

Proteostasis unbalance of nucleophosmin 1 in Acute Myeloid Leukemia: An aggregomic perspective

The role exerted by the nucleus in the regulation of proteostasis in both health and disease is recognized of outmost importance, even though not fully understood. Many recent investigations are focused on its ability to modulate and coordinate protein quality control machineries in mammalian cells. Nucleophosmin 1 (NPM1) is one of the most abundant nucleolar proteins and its gene is mutated in ~30% of Acute Myeloid Leukemia (AML) patients.Mutations are localized in the C-terminal domain of the protein and cause cytoplasmatically delocalized and possibly aggregated forms of NPM1 (NPM1c+). Therapeutic interventions targeted on NPM1c+ are in demand and, to this end, deeper knowledge of NPM1c+ behavior in the blasts' cytosol is required.Here by means of complementary biophysical techniques we compared the conformational and aggregative behavior of the entire C-terminal domains of NPM1wt and type A NPM1c+ (bearing the most common mutation). Overall data show that only Cterm_mutA is able to form amyloid-like assemblies with fibrillar morphology and that the oligomers are toxic in human neuroblastoma SHSY cells. This study adds a novel piece of knowledge to the comprehension of the molecular roles exerted by cytoplasmatic NPM1c+ and suggests the exploitation of the amyloidogenic propensity of NPM1c+ as a new strategy for targeting AML with NPM1 mutations.

Flow cytometry in detection of Nucleophosmin 1 mutation in acute myeloid leukemia patients: A reproducible tertiary hospital experience.

Nucleophosmin 1 (NPM1) mutation is one of the most frequent gene mutations in adult acute myeloid leukemia (AML), being detected in 35% of all cases and in up to 60% of patients with normal karyotype AML. AML with mutated NPM1 has distinct pathology, immunophenotyping, and confirmed favorable prognostic significance. Hence, AML with mutated NPM1 is a separate entity in the revised 2016 World Health Organization classification. This study aimed to evaluate the use of a reproducible flow cytometry approach in the assay of mutant NPM1 protein in AML patients and to correlate flow cytometric results with the NPM1 gene mutation. Eighty-nine newly diagnosed AML patients were evaluated for the expression of mutant NPM1 using flow cytometry and for the presence of NPM1 exon 12 mutations using high-resolution melting polymerase chain reaction (HRM PCR). The NPM1 mutation was found in 35 (39.3%) patients by HRM PCR. These patients showed a significantly higher level of percentage of positive-stained cells (% positive cells) and normalized median fluorescence intensity (MFI) for mutant NPM1 by flow cytometry than the negative mutation group. Flow cytometric detection of mutant NPM1 offers a possible tool to indicate NPM1 mutational status.

Abstract 1920: Response to hypomethylating agents based on cytidine deaminase expression, genetic polymorphisms, and NPM1 mutation status in acute myeloid leukemia

Abstract Response to hypomethylating agents (HMAs) in patients (pts) with acute myeloid leukemia (AML) is variable. Data on biological predictors of response is limited. Cytidine deaminase (CDA) inactivates HMAs. Increased CDA activity may lead to HMA resistance. Using core pathway analysis, we identified nucleophosmin 1 (NPM1) indirectly influences CDA expression. We hypothesized that responses to HMAs occur in the setting of decreased CDA expression regulated by NPM1 and investigated the relationship between NPM1 status, CDA expression, single nucleotide polymorphisms (SNPs) in CDA, and HMA response in pts with AML. AML pts with banked samples who received HMA-based therapy between 1/2014 to 12/2018 were reviewed. Responses following at least 2 cycles of HMA were categorized as responders (R) or non-responders (NR). Pt, disease, NPM1 status, and treatment characteristics were summarized. CDA gene and protein expression was examined in bone marrow and peripheral blood samples at diagnosis using qRT-PCR and CDA sandwich ELISA, respectively. CDA gene expression levels were normalized to the housekeeping gene, 18s, and the comparative CT method was used to assess expression. Comparisons based on response and NPM1 mutation status were performed using the Mann-Whitney U test. 17 SNPs previously shown to alter CDA activity were selected for analysis. SNPs were determined using real-time PCR with allele specific probes; longer insertions/deletions were identified by sanger sequencing. Univariate logistic regression analysis was performed to discern the association between SNPs in CDA and response to HMAs. 54 pts had available blood, marrow, or buccal samples available for analysis. 33 pts provided blood or marrow samples for gene and protein analysis prior to HMA. 22 pts (67%) were classified as R in this cohort. 35 pts had available buccal swabs for genotyping, and 28 pts (80%) were classified as R. Median OS was 21 months (mo) for all pts, 23 mo among R, and 18 mo in NR. CDA expression was significantly decreased in NPM1 wild type pts compared to NPM1 mutant pts (p=0.02) but did not differ in R compared to NR. No significant differences were identified in CDA protein expression based on NPM1 status or response. No SNPs were significantly associated with response. Baseline CDA gene expression in bulk tumor cells was significantly lower in NPM1 wild type pts compared to NPM1 mutant pts but no different between pts responding to HMAs compared to NR. There was no clear correlation between CDA protein expression and NPM1 status or response. None of the CDA SNPs were predictive of response to HMAs. This analysis reveals feasibility of assessing CDA activity in this population. Our small sample size limits our ability to determine CDA activity as a biological predictor of response, and ongoing pt accrual will allow for further exploration of the role of CDA in HMA response. Citation Format: Brittany Knick Ragon, Issam S. Hamadeh, C Greer Vestal, Alicia Hamilton, Mathew L. Smith, Danielle Boselli, Jing Ai, Thomas G. Knight, Michael R. Grunwald, Jonathan M. Gerber, Edward A. Copelan, Lawrence J. Druhan, Belinda R. Avalos, Nury M. Steuerwald, Jai N. Patel. Response to hypomethylating agents based on cytidine deaminase expression, genetic polymorphisms, and NPM1 mutation status in acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1920.

NPM1 Biology in Myeloid Neoplasia

Nucleophosmin (NPM1) mutations are encountered in myeloid neoplasia and are present in ~ 30% of de novo acute myeloid leukemia cases. This review summarizes features of mutant NPM1-related disease, with a particular emphasis on recent discoveries relevant to disease monitoring, prognostication, and therapeutic intervention. Recent studies have shown that HOX/MEIS gene overexpression is central to the survival of NPM1-mutated cells. Two distinct classes of small molecule drugs, BH3 mimetics and menin-MLL interaction inhibitors, have demonstrated exquisite leukemic cell toxicity in preclinical AML models associated with HOX/MEIS overexpression, and the former of these has shown efficacy in older treatment-naïve NPM1-mutated AML patients. The results of ongoing clinical trials further investigating these compounds will be of particular importance and may alter the clinical management of patients with NPM1-mutated myeloid neoplasms. Significant scientific advancements over the last decade, including improved sequencing and disease monitoring techniques, have fostered a much deeper understanding of mutant NPM1 disease biology, prognostication, and opportunities for therapeutic intervention. These discoveries have led to the development of clinical assays that permit the detection and monitoring of mutant NPM1 and have paved the way for future investigation of targeted therapeutics using emerging cutting-edge techniques.

Outcomes of older patients with NPM1-mutated AML: current treatments and the promise of venetoclax-based regimens

Nucleophosmin-1 mutations (NPM1+) occur in ∼30% of acute myeloid leukemia (AML) patients. Although typically associated with favorable prognosis, the beneficial impact of NPM1+ decreases with increasing age in patients treated with standard intensive chemotherapy (IC) or hypomethylating agents (HMAs). This retrospective analysis compared outcomes of NPM1+ AML patients treated with 1 of 3 induction approaches: HMA plus BCL-2 inhibitor venetoclax (VEN), HMA, or IC therapy. Composite complete response (CRc: CR + CR with incomplete count recovery) was seen in 96% (27/28), 36% (17/47), and 89% (204/228) of HMA + VEN, HMA, and IC patients, respectively (HMA + VEN vs HMA, P < .001; HMA + VEN vs IC, P = .10). Older patients (age >65 years) treated with HMA + VEN, HMA, or IC had CR rates of 88%, 28%, and 56%, respectively (HMA + VEN vs HMA, P < .001; HMA + VEN vs IC, P = .01). Significant improvement in overall survival (OS) was seen in patients age >65 years treated with HMA + VEN vs HMA (not reached [NR] vs 0.4 years; P < .001) or IC (NR vs 0.93 years; P = .001). Older patients treated with HMA + VEN had OS of 80% after median 1-year follow-up, with estimated 2-year OS of 70%. In the multivariable Cox model analysis, HMA + VEN was associated with a 69% lower risk of death compared with IC (hazard ratio, 0.31; 95% confidence interval, 0.12-0.83; type I error-adjusted P = .038). HMA + VEN combinations demonstrated impressive results compared with traditional standard-of-care regimens in older patients with NPM1+ AML.

The prognostic impact of FLT3-ITD and NPM1 mutation in adult AML is age-dependent in the population-based setting

AbstractIn acute myeloid leukemia (AML) FLT3 internal tandem duplication (ITD) and nucleophosmin 1 (NPM1) mutations provide prognostic information with clinical relevance through choice of treatment, but the effect of age and sex on these molecular markers has not been evaluated. The Swedish AML Registry contains data on FLT3-ITD and NPM1 mutations dating to 2007, and 1570 adult patients younger than 75 years, excluding acute promyelocytic leukemia, had molecular results reported. Females more often had FLT3ITD and/or NPM1mut (FLT3ITD: female, 29%; male, 22% [P = .0015]; NPM1mut: female, 36%; male, 27% [P = .0001]), and more males were double negative (female, 53%; male, 64%; P < .0001). Patients with FLT3ITD were younger than those without (59 vs 62 years; P = .023), in contrast to patients with NPM1mut (62 vs 60 years; P = .059). Interestingly, their prognostic effect had a strong dependence on age: FLT3ITD indicated poor survival in younger patients (<60 years; P = .00003), but had no effect in older patients (60-74 years; P = .5), whereas NPM1mut indicated better survival in older patients (P = .00002), but not in younger patients (P = .95). In FLT3ITD/NPM1mut patients, the survival was less dependent on age than in the other molecular subsets. These findings are likely to have clinical relevance for risk grouping, study design, and choice of therapy.

Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

Nucleophosmin mutations confer an independent favorable prognostic impact in 869 pediatric patients with acute myeloid leukemia

Studies on the clinical significance of Nucleophosmin (NPM1) mutations in pediatric AML in a large cohort are lacking. Moreover, the prognosis of patients with co-occurring NPM1 and FLT3/ITD mutations is controversial. Here, we analyzed the impact of NPM1 mutations on prognoses of 869 pediatric AML patients from the TAGET dataset. The frequency of NPM1 mutations was 7.6%. NPM1 mutations were significantly associated with older age (P < 0.001), normal cytogenetics (P < 0.001), FLT3/ITD mutations (P < 0.001), and high complete remission induction rates (P < 0.05). Overall, NPM1-mutated patients had a significantly better 5-year EFS (P = 0.001) and OS (P = 0.016) compared to NPM1 wild-type patients, and this favorable impact was maintained even in the presence of FLT3/ITD mutations. Stem cell transplantation had no significant effect on the survival of patients with both NPM1 and FLT3/ITD mutations. Multivariate analysis revealed that NPM1 mutations were independent predictors of better outcome in terms of EFS (P = 0.004) and OS (P = 0.012). Our findings showed that NPM1 mutations confer an independent favorable prognostic impact in pediatric AML despite of FLT3/ITD mutations. In addition, pediatric AML patients with both NPM1 and FLT3/ITD mutations appear to have favorable prognoses and may not need hematopoietic stem cell transplantations.

Nucleophosmin1 (NPM1) abnormality in hematologic malignancies, and therapeutic targeting of mutant NPM1 in acute myeloid leukemia.

Nucleophosmin (NPM1) is an abundant nucleolar protein that is implicated in a variety of biological processes and in the pathogenesis of several human malignancies. For hematologic malignancies, approximately one-third of anaplastic large-cell non-Hodgkin’s lymphomas were found to express a fusion between NPM1 and the catalytic domain of anaplastic lymphoma receptor tyrosine kinase. About 50–60% of acute myeloid leukemia patients with normal karyotype carry NPM1 mutations, which are characterized by cytoplasmic dislocation of the NPM1 protein. Nevertheless, NPM1 is overexpressed in various hematologic and solid tumor malignancies. NPM1 overexpression is considered a prognostic marker of recurrence and progression of cancer. Thus, NPM1 abnormalities play a critical role in several types of hematologic malignancies. This has led to intense interest in the development of an NPM1 targeting strategy for cancer therapy. The aim of this review is to summarize present knowledge on NPM1 origin, pathogenesis, and therapeutic interventions in hematologic malignancies.

Nucleophosmin Gene Mutation in Acute Myeloblastic Leukemia: Apoptotic Role

Introduction and objectives: Since Falini and his co-workers reported the expression of nucleophosmin (NPM) gene mutation in acute myeloblastic leukemia (AML), and various research in succession clarified the role of mNPM in AML. We previously studied the proliferative role of mNPM in AML in a trial to discover its role in AML development and leukemogenesis. This study aimed to complete the scenario and investigate the apoptotic role of mNPM AML. Another objective was to assess the effect of NPM mutational state on response to etoposide treatment in AML. Materials and methods: In this study human leukemia cell lines, HL60 and OCI-AML3 were used as models for AMLs bearing wild type (wt) and mutated (m) NPM, respectively. The study was conducted by using viability studies. The obtained results were reaffirmed by immunocytochemical and immunoblotting analyses. Results were interpreted and presented with the appropriate computer softwares. Results and conclusions: Interpretation of data showed normal growth and delayed apoptotic response of etoposide treated cells bearing mNPM as compared with cells carrying wtNPM and the control. Also we noted irreversible cytoplasmic translocation of NPM that was dependent on the duration and extent of the etoposide induced cytotoxicity in cells with wtNPM. Proteomic analysis of NPM revealed that protein expression in the etoposid lysates was approximately similar to the untreated controls We concluded that wtNPM has a pro-apoptotic effect while mNPM has an anti-apoptotic effect, suggested that therapeutic response to etoposide treatment in AML could be variable from one patient to another depending on the molecular basis of leukemia development in each case. Also, it was concluded that AML cells bearing nucleophosmin mutation are resistant to etoposide effect.

Molecular roulette: nucleophosmin mutations in AML are orchestrated through N-nucleotide addition by TdT

AbstractNucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML). AML with mutated NPM1 is recognized as a separate entity in the World Health Organization 2016 classification and carries a relatively favorable prognosis. NPM1 mutations are predominantly 4-bp duplications or insertions in the terminal exon that arise through an unknown mechanism. Here we analyze 2430 NPM1 mutations from 2329 adult and 101 pediatric patients to address their origin. We show that NPM1 mutations display the hallmarks of replication slippage, but lack suitable germline microhomology available for priming. Insertion mutations display G/C-rich N-nucleotide tracts, with a significant bias toward polypurine and polypyrimidine stacking (P < .001). These features suggest terminal deoxynucleotidyl transferase (TdT) primes replication slippage through N-nucleotide addition, with longer syntheses manifesting as N-regions. The recurrent type A, type D, and type B mutations require 1, 2, and 3 N-nucleotide extensions of T, CC, and CAT, respectively, with the last nucleotide used as occult microhomology. This TdT-mutator model successfully predicts the relative incidence of the 256 potential 4-bp insertion/duplication mutations at position c.863_864 over 4 orders of magnitude (ρ = 0.484, P < .0001). Children have a different NPM1 mutation spectrum to adults, including a shift away from type A mutations and toward longer N-regions, consistent with higher TdT activity in pediatric myeloid stem cells. These findings complement our FLT3-ITD data, suggesting illegitimate TdT activity contributes to around one-half of AMLs. AML may therefore reflect the price for adaptive immunity.

DNMT3Awt NPM1-Mutation Defines a Subgroup of MDS with Special Favorable Outcomes Towards Decitabine Therapy

Hypomethylating agents (HMA) (including decitabine and azacitidine) are considered standard of care for higher risk myelodysplastic syndrome (MDS). Clinical data showed only about 30% cases achieved complete response (CR) by decitabine. Mutations in the nucleophosmin-1 (NPM1) gene is one of the most common somatic mutations identified in de novo acute myeloid leukemia (AML) and have also been found in 1% to 5% of MDS patients although with different mutation locus (L287fs) when compared to that in AML (W288fs). Till now, the response and survival of NPM1-mutated MDS patients treated with HMA remains unknown. Here, we retrospectively analyzed higher risk MDSs who accepted decitabine therapy in our center. From December 2009 to July 2018, a total of 194 patients received decitabine induction treatment by 20mg/m2 intravenously for 5 consecutive days every 4-6 weeks. The median therapy course was four. Among them, twelve patients (6.2%) harbored NPM1/L287fs mutation. The median decitabine therapy cycle for the 12 NPM1 mutated patients was also four. To our interest, patients harboring NPM1 mutations achieved a relatively higher CR rate (6 of 12 cases, 50%) when compared to that of patients without NPM1 mutations (59 of 182 cases, 32.4%) , although without statistical significance (p = 0.304). Moreover, when the most common co-mutated genes DNMT3A (6 of 12 cases, 50%) (Figure 1a) was ruled out, patients harboring NPM1 mutation (DNMT3A wild type) achieved a CR rate of 83.3% (five of six), which is significantly higher than that of patients without NPM1 mutation (p = 0.018) (Figure 1b). Of note, when paired sequencing were analyzed, six patients who achieved CR by decitabine showed loss of mutated NPM1; One patients who achieved hematological improvement (HI) showed decreased variant allele frequency (VAF) of NPM1 mutation; Whereas two patients with no response (NR) showed unchanged NPM1 mutation (Figure 1d). Notably, a prolonged relapse-free survival (PFS) was observed in CR patients with NPM1 mutation and DNMT3A wild type (NPM1mut DNMT3Awt) even without any subsequent therapies after receiving 4-5 cycles of decitabine (Figure 1c). The median RFS of CR patients with NPM1mut DNMT3Awt was 66 months, which is significantly longer than that in patients without NPM1mutation (13.5M, p = 0.006) (Figure 1e). A remarkably prolonged median survival was also shown in patients harboring NPM1mut DNMT3Awt (median survival of 80M), which is significantly longer than that of patients without NPM1 mutation (18M) (p = 0.012) (Figure 1f). Interestingly, except with DNMT3A and PTPRD co-mutations, the response and survival of patients harboring NPM1 mutations treated with decitabine were favorable even when co-mutated with IDH2, NRAS, FLT3. In conclusion, NPM1 mutation with DNMT3A wild type defines a specific subgroup of MDS with a good response and prolonged survival by decitabine therapy, even when they were with some prognosis-poor co-mutations and without subsequent treatment. Enlarged sample of randomized controlled studies are needed to confirm our preliminary findings. Figure 1 Disclosures No relevant conflicts of interest to declare.

Clinical Validation and Implementation of a Measurable Residual Disease Assay for NPM1 in Acute Myeloid Leukemia by Error-Corrected Next-Generation Sequencing.

Nucleophosmin 1 (NPM1) is one of the most commonly mutated genes in acute myeloid leukemia, with mutations observed in approximately 30% of all adult cases. The persistence of NPM1 mutations following chemotherapy is associated with a greater risk of relapse as well as a lower rate of survival, making NPM1 measurable residual disease (MRD) an informative clinical target. Herein, we have developed a straightforward unique molecular identifier (UMI)-based amplicon next-generation sequencing method for the detection of NPM1-mutated MRD that addresses some of the limitations present in other assays. The NPM1 assay allowed for accurate counting of individual mutant and wild-type molecules down to 0.01% variant allelic frequency. In silico contamination experiments highlighted the ability of this UMI methodology to maximize specificity through dramatic reductions in sequencing/demultiplexing bleed-through error. Performance and clinical utility of the NPM1 MRD assay are established via both validation experiments and analyses of live performance over 1.5years of routine clinical service.

Elevated HOX gene expression in acute myeloid leukemia is associated with NPM1 mutations and poor survival

Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic progenitor cells and the most common malignant myeloid disorder in adults. Several gene mutations such as in NPM1 (nucleophosmin 1) are involved in the pathogenesis and progression of AML. The aim of this study was to identify genes whose expression is associated with driver mutations and survival outcome. Genotype data (somatic mutations) and gene expression data including RNA-seq, microarray, and qPCR data were used for the analysis. Multiple datasets were utilized as training sets (GSE6891, TCGA, and GSE1159). A new clinical sample cohort (Semmelweis set) was established for in vitro validation. Wilcoxon analysis was used to identify genes with expression alterations between the mutant and wild type samples. Cox regression analysis was performed to examine the association between gene expression and survival outcome. Data analysis was performed in the R statistical environment. Eighty-five genes were identified with significantly altered expression when comparing NPM1 mutant and wild type patient groups in the GSE6891 set. Additional training sets were used as a filter to condense the six most significant genes associated with NPM1 mutations. Then, the expression changes of these six genes were confirmed in the Semmelweis set: HOXA5 (P = 3.06E−12, FC = 8.3), HOXA10 (P = 2.44E−09, FC = 3.3), HOXB5 (P = 1.86E−13, FC = 37), MEIS1 (P = 9.82E−10, FC = 4.4), PBX3 (P = 1.03E−13, FC = 5.4) and ITM2A (P = 0.004, FC = 0.4). Cox regression analysis showed that higher expression of these genes – with the exception of ITM2A – was associated with worse overall survival. Higher expression of the HOX genes was identified in tumors harboring NPM1 gene mutations by computationally linking genotype and gene expression. In vitro validation of these genes supports their potential therapeutic application in AML.

Identification of FLT3 and NPM1 Mutations in Patients with Acute Myeloid Leukaemia.

Objective:The most frequent acquired molecular abnormalities and important prognostic indicators in patients with Acute Myeloid Leukaemia (AML) are fms-like tyrosine kinase-3 gene (FLT3) and nucleophosmin-1 (NPM1) mutations. Our study aims to develop a cost effective and comprehensive in-house conventional PCR method for detection of FLT3-ITD, FLT3-D835 and NPM1 mutations and to evaluate the frequency of these mutations in patients with cytogenetically normal (CN) AML in our population. Methods:A total of 199 samples from AML patients (95 women, 104 men) were included in the study. Mutation analyses were performed using polymerase chain reaction (PCR) and gene sequencing. Result:Sixty-eight patients were positive for the mutations. FLT3-ITD mutations were detected in 32 patients (16.1%), followed by FLT3-D835 in 5 (2.5%) and NPM1 in 54 (27.1%). Double mutations of NPM1 and FLT3-ITD were detected in 23 cases (11.6%). Assays validation were performed using Sanger sequencing and showed 100% concordance with in house method. Conclusion:The optimized in-house PCR assays for the detection of FLT3-ITD, FLT3-D835 and NPM1 mutations in AML patients were robust, less labour intensive and cost effective. These assays can be used as diagnostic tools for mutation detection in AML patients since identification of these mutations are important for prognostication and optimization of patient care.

Investigation of screening method for DNMT3A mutations by high‐resolution melting analysis in acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease associated with various genetic abnormalities. Somatic mutations in nucleophosmin 1 (NPM1), fms-related tyrosine kinase 3 (FLT3), and DNA methyltransferase 3 alpha (DNMT3A) are the most frequent mutations associated with AML. However, because DNMT3A mutations are broadly distributed, they are challenging to analyze in routine laboratory tests. Hence, we developed a rapid screening method for DNMT3A mutations by high-resolution melting (HRM) analysis for clinical use at the point of AML diagnosis. The detection limit for DNMT3A mutations from exons 8-23 by an HRM analysis was investigated using plasmid mixtures. In 69 patients with AML, somatic mutations in NPM1, FLT3-internal tandem duplication (ITD), FLT3-tyrosine kinase domain (TKD), DNMT3A, and isocitrate dehydrogenase 1/2 were screened using HRM analysis, and direct sequencing was performed for positive samples. High-resolution melting analysis enabled complete mutation detection in samples with 20% mutant alleles in all regions. In a clinical laboratory test, DNMT3A mutations were detected in 12 cases (17.3%), and we identified five novel mutations. Simultaneous NPM1, FLT3-ITD, and DNMT3A mutations constituted the most common pattern (30%) in de novo cytogenetically normal AML. High-resolution melting analysis has sufficient performance for the detection of DNMT3A mutations in AML. This approach can facilitate rapid AML genotyping at diagnosis in clinical settings.

Survival outcomes in patients with good risk acute myeloid leukemia: A single-center retrospective review.

e18519 Background: Cytogenetics and gene mutations play a significant role in determining necessity of allogeneic transplant in patients with AML. Core binding cytogenetic factors (CBF) such as t(8;21), inv 16, and t(16;16) as well as molecular mutations in nucleophosmin-1 (NPM1) and bi-allelic CCAAT/enhancer binding protein alpha (CEBPA) mutations in cytogenetically normal patients have conferred improved relapse-free survival (RFS) and overall survival (OS) rates. We report outcomes of these “good-risk” patients in an Appalachian population, to evaluate differences from previous reports. Methods: A total of 79 patients with AML diagnosed between July 2007 and July 2017 at West Virginia University (WVU) who qualified for good-risk disease per ELN 2017 criteria were retrospectively analyzed for RFS and OS. Eighteen of these patients were excluded from our final analysis: 10 for primary refractory disease and 8 for proceeding to transplant in first remission. Results: A total of 61 patients were included in our analysis, with a median age of 56 (range 11-81), 32 females, and 29 males. Good-risk features included 29 patients with NPM1, no patients with CEBPA, 19 with inv 16, 9 with t(8;21), and 1 with t(16;16). All patients received cytarabine and an anthracycline induction chemotherapy, and a median of 4 cycles (range 1 -4) of high-dose cytarabine consolidation chemotherapy. The RFS and OS at 4 years was 36% and 50%, respectively. There were no occurrences of treatment-related mortality among the patients. Among the 22 patients relapsing, 12 (55%) were able to achieve a second remission and received allogeneic transplant. Conclusions: Our outcomes appear somewhat lower than previous reports, but continuing to highlight the ability to achieve long-term remissions in this subset of patients, without the need for allogeneic transplant. Continued efforts should focus on improving outcomes in these patients, to further prevent relapses.