NPM1 Gene Mutations Research Articles

Expression and Clinical Significance of CD73 in Acute Myeloid Leukemia Patients with NPM1 Mutation

To investigate the expression of CD73 in acute myeloid leukemia (AML) patients with NPM1 mutant and wild-type, and to evaluate the therapeutic efficacy and prognosis of CD73 to the AML patients. 160 patients with AML treated in our hospital from June 2015 to June 2019 were enrolled, and 40 non-AML bone marrow samples from healthy people were selected as controls during the same period. The expression of CD73 in healthy people, NPM1 mutation and NPM1 wild-type AML patients were compared, and the relationship between the expression of CD73 and its clinicopathological characteristics, as while as efficacy in AML patients were analyzed. The patients were followed up, and the influence of CD73 to the prognosis of different AML patients was analyzed. The positive expression rate of CD73 in AML patients (23.75%) was significantly higher than that in the healthy control group (0.62%), and the positive expression rate of CD73 in AML patients with NPM1 mutation (74.75%) was significantly higher than that with NPM1 wild-type (25.51%) (both P<0.001). AML patients with CD73 positive expression was associated with age, FAB typing, disease risk classification, and NPM1 gene mutation (both P<0.05). The overall survival rate of AML patients with NPM1 gene mutation was 75.98%, which was significantly higher than the patients with NPM1 wild-type (34.68%)(P<0.001), the median survival time of AML patients with NPM1 gene mutation in the CD73+ group was 21 months, which was significantly longer than the patients in the CD73- group (11 months)(P<0.001), the median survival time of AML patients with NPM1 wild-type in the CD73+ group was 13 months, which was significantly shorter than the patients in the CD73- group (18 months) (P<0.001). The expression of CD73 was increased in AML patients with NPM1 gene mutation, and CD73 showed different prognostic significance in AML patients with different NPM1 gene mutation. The combination of clinicopathologic features, CD73 expression and NPM1 gene in AML patients is helpful to determine their prognosis and guide the formulation of relevant treatment plans.

Methodological Research on Rapid Detection and Genotyping of NPM1 Gene Mutations in Leukemia

To establish a method for rapid detection and typing of NPM1 mutations in acute myeloid leukemia (AML) by fluorescence melting curve analysis technology. A pair of primers and a fluorescent single-stranded probe (molecule beacon) were designed for the mutant genes mutA, mutB, mutD in exon 12 of nucleopsin (NPM1) and wild type. With a real-time qPCR, the A, B, and D gene mutations of NPM1 were detected and typed by different-melting curve peak value of the probe through RT-PCR. This method could detected the mutations of A, B, and D in NPM1 effectively with a sensitivity of 1%. Furthermore, 62 AML clinical samples were evaluated by the method. In the results, the detection rate and typing of NPM1 mutations were consistent with the sequencing results of clinical samples. There are three features in the method of fluorescence melting curve analysis: stable PCR system, easy to operate, and the easily distinguishable results. The method might meet the demand for rapid typing of NPM1 gene mutation in early diagnosis or concomitant diagnosis of AML.

Study of the Age Dynamics of Morphological Subtypes of Acute Myeloid Leukemia in Adults

Aim: to determine the age dynamics of morphological subtypes of acute myeloid leukemia in adult patients. Materials and methods. The study group consisted of 132 patients (including 34 aged 15 to 45 years, 46 aged 45-60 years, 52 aged over 60 years) with newly diagnosed AML. Patients with acute promyelocytic leukemia were not included in the study. The diagnosis was made in accordance with WHO recommendations and FAB classification criteria. In all cases, morphological verification was performed, including cytological, cytochemical studies and immunophenotyping. Detection of chromosomal abnormalities was performed using standard cytogenetic and real-time polymerase chain reaction methods. Point mutations were screened in 7 genes: c-KIT, DNMT3A, FLT3, NRAS, NPM1, TP53 and WT1 by direct sequencing method. Results. The distribution of morphological variants of AML according to the FAB classification was as follows: M2 — 47.7%, M4 — 26.5%, M1 — 7.6%, M0 — 4.5%, M6 — 4.5%, M5 — 3.0%, M4eo — 2.3%, blastic plasmacytoid dendritic cell neoplasm — 1.5%, acute myelofibrosis — 0.8%, acute hybrid leukemia — 0.8%. Complex genetic abnormalities were detected in 18.2%, specific chromosome abnormalities associated with a favorable prognosis — 7.6%, specific chromosome abnormalities associated with an unfavorable prognosis — 5.3%, normal cytogenetics — 28.8%. Mutations in the FLT3 gene were detected in 15.0%, NPM1 — 14.3%, DNMT3A — 12.5%, NRAS — 12.2%, TP53 — 9.6%, c-KIT — 5.7%, WT1 — 4.2%. It was revealed that the frequency of acute myeloid leukemias with monocytic differentiation (M4, M4eo and M5 according to FAB) had a statistically significant tendency to decrease with increasing age: in the subgroup of patients younger than 45 years, it was 44.1%, in the age of 45-60 years — 32.6%, in the age of 60 years and older — 23.1%. These differences correlated with the age-related dynamics of the frequency of genetic abnormalities in AML, including a decrease in the frequency of NPM1 gene mutations and specific chromosomal aberrations, as well as an increase in the frequency of TP53 abnormalities in older age groups.

Prognostic Value of CD123 in Acute Myeloid Leukemia Patients with Intermediate Risk in Normal Karyotype

To investigate the expression of CD123 in patients with acute myeloid leukemia (AML) and its relationship between clinical features, concomitant fusion gene or gene mutation, efficacy and prognosis. 365 patients with newly diagnosed AML (except M3) treated in the First Affiliated Hospital of Zhengzhou University were enrolled and retrospective analysis, and multi-parameter flow cytometry was performed to detect the expression of CD123 in myeloid leukemia cell population. CD123≥20% was defined as positive. Clinical features, concomitant fusion gene or gene mutation, efficacy and prognosis of CD123+ and CD123- patients were compared and analyzed. The positive rate of CD123 in 365 newly diagnosed AML patients was 38.9%. Compared with the CD123- group, the white blood cell count (WBC), lactate dehydrogenase (LDH) level and bone marrow blast cell ratio were higher in the CD123+ group, and the ratio of NPM1 and DMNT3a gene mutations and the CBFβ-MYH11 fusion gene was significantly increased, while the ratio of CEBPA gene mutation and AML-ETO fusion gene was significantly reduced. The rate of first inducing complete remission and total remission in CD123+ group was significantly lower than that in CD123- group. There was no significant difference in recurrence rate between the two groups (P>0.05). Survival analysis showed that in 77 AML patients with normal karyotype and intermediate risk, the 2-year overall survival (OS) rate and event-free survival (EFS) rate of the CD123+ group were significantly lower than those of the CD123- group. Multivariate analysis showed that CD123 was an independent prognostic risk factor for OS and EFS in AML patients with normal karyotype and intermediate risk. CD123 positive indicates that AML patients have higher tumor burden and are more difficult to reach remission. It is an independent risk factor for OS and EFS in patients with normal karyotype and intermediate risk, which is important to evaluate the prognosis of patients with AML without specific prognostic marker.

Assay Specificity and Sensitivity Evaluation of Cepheid GeneXpert® NPM1 Prototype for Mutation Detection in Acute Myeloid Leukemia

Background: Mutations in thenucleophosmin1 (NPM1) are considered as founder mutations in acute myeloid leukemia (AML). NPM1 mutations in exon 12 occur in about 60% adult AML patients with normal karyotype. Among all NPM1 subtypes, type A (69.15%), type B (7.41%) and type D (7.41%) are found to be most frequent. The evaluation of minimal residual disease (MRD) has been considered critical to more precisely define AML response to clinical treatment, thereby refining disease risk stratification. Currently, AML with NPM1 gene mutations is recognized as a distinct entity, due to its unique clinical features. A large body of evidence demonstrated a significant independent prognosis factor of molecular MRD monitoring in NPM1-mutated AML. Objectives: Here we present GeneXpert NPM1 prototype, a fast and promising test for use in AML mutation detection (this prototype is for research use only, not for use in diagnostic procedures, and not reviewed by any regulatory body). NPM1 prototype is an easy-to-use-sample in/results out, and highly sensitive assay. The turnaround time is as fast as less than 3 hours. NPM1 prototype is designed for the quantitative detection of NPM1 mutant mRNA transcripts (types A, B and D in exon 12) and the ABL endogenous control mRNA transcript in peripheral blood of AML patients. This test is also designed for use as an aid in the monitoring of mutant NPM1 mRNA transcript levels in patients diagnosed with NPM1-mutated AML. This test does not differentiate between A, B or D type mutant NPM1 transcripts and does not detect or monitor other rare types of mutant NPM1. Results: Through assay optimization, we were able to reach limit of detection (LoD) within 0.05% for three NPM1 subtypes among all screened EDTA donor blood samples. Ct-copy number regression curve aligns well among three subtypes, within detection range. Our assay specificity has been above 95% among 30 screened EDTA donor blood samples. Conclusion: In summary, GeneXpert NPM1 prototype shows promise as a fast test for use in AML mutation detection.The presented NPM1 prototype has a great advantage covering most prevalent NPM1 mutation types up to 94% of all NPM1 subtypes. Our results demonstrated that our prototype shows satisfactory assay specificity and sensitivity. Clinical samples will be included for our clinical validation in the future. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia with mutated NPM1 mimics acute promyelocytic leukemia presentation.

To investigate clinicopathological and molecular features of NPM1-mutated acute myeloid leukemia that presented with infrequent acute promyelocytic leukemia (APL)-like phenotype and clinical presentation. Cases with both de novo or secondary Acute Myeloid Leukemia (AML) were retrieved. Data from flow cytometry immunophenotyping, cytogenetics, molecular studies, and clinical presentation were analyzed. Cases presented with abnormal coagulation parameters and low platelets count; four of them showed a DIC index compatible with overt DIC. Two cases showed Auer rods. In all cases, immunophenotypes mimicked APL: blasts expressed CD33, CD13, and cytoplasmic MPO but did not express CD34, HLA-DR, or CD11b. Notably, CD4 expression was observed in all cases. Neither t(15;17) nor PML/RARα gene rearrangement was detected. NPM1 gene mutation was identified in all cases. In four cases, TET2 or IDH2 co-mutations were identified. Our findings provide additional evidence of association between NPM1-mutated AML with TET2 or IDH2 co-mutations and the APL-like immunophenotype. This AML subset was found to exist in both de novo and secondary AML. High WBC count and blasts with low to moderate side scatter and significant expression of CD4 are observed features that could assist in the differential diagnosis with APL. The occurrence of significant elevated D-dimer levels, or even overt DIC observed at diagnosis in these cases could be relevant for this AML subtype.

Correlation of Early Minimal Residual Disease Level with Prognosis in AML Patients with NPM1 Mutation Positive after Chemotherapy

To analyze the correlation of the minimal residual disease level with the prognosis of the AML patients with NPM1 gene mutation positive after chemotherapy. The clinical data of 112 newly diagnosed adult AML patients with positive NPM1 gene were collected and retrospectively analyzed. The correlation of the transcripts of NPM1 gene mutation with prognosis of patients was analyzed. In 112 AML patients, the median transcript level of NPM1 gene mutation accounted for 83.68% (5.86%-486.57%), FLT3-ITD mutation positive was found in 44 cases (39.29%), chromosomal abnormalities in 22 cases (19.64%) and complete remission in 96 cases (85.71%). Multivariate logistic regression analysis showed that the initial induction therapy and white blood cell count closely related with complete remission (P＜0.05). The median follow-up time was 22 (3-36) months, and the 3-year overall survival rate was 66.07% in 112 patients. Multivariate logistic regression analysis showed that both the high level of minimal residual disease at the initial complete remission and the high level of minimal residual disease after consolidation therapy were the independent risk factors for overall survival (P＜0.05). In newly diagnosed adult AML patients with NPM1 mutation positive, the early high level of minimal residual disease after chemotherapy closely relates with poor prognosis.

Correlation of Early Minimal Residual Disease Level with Prognosis in AML Patients with NPM1 Mutation Positive after Chemotherapy

To analyze the correlation of the minimal residual disease level with the prognosis of the AML patients with NPM1 gene mutation positive after chemotherapy. The clinical data of 112 newly diagnosed adult AML patients with positive NPM1 gene were collected and retrospectively analyzed. The correlation of the transcripts of NPM1 gene mutation with prognosis of patients was analyzed. In 112 AML patients, the median transcript level of NPM1 gene mutation accounted for 83.68% (5.86%-486.57%), FLT3-ITD mutation positive was found in 44 cases (39.29%), chromosomal abnormalities in 22 cases (19.64%) and complete remission in 96 cases (85.71%). Multivariate logistic regression analysis showed that the initial induction therapy and white blood cell count closely related with complete remission (P＜0.05). The median follow-up time was 22 (3-36) months, and the 3-year overall survival rate was 66.07% in 112 patients. Multivariate logistic regression analysis showed that both the high level of minimal residual disease at the initial complete remission and the high level of minimal residual disease after consolidation therapy were the independent risk factors for overall survival (P＜0.05). In newly diagnosed adult AML patients with NPM1 mutation positive, the early high level of minimal residual disease after chemotherapy closely relates with poor prognosis.

Hyperleucocytosis grading score and NPM1 gene mutation among patients with acute myeloid leukemia: Malaysian experience

This is the first study evaluating the frequencies of nucleophosmin (NPM1) gene mutation according to the hyperleukocytosis (HL) grading score. The current two cut off values of this score in acute myeloid leukaemia (AML) patients are [1] those with 50–100 × 109/L and [2] those with ≥ 100 × 109/L total WBC count. The score represents the current clinically based definition of HL in patients with AML. A total of 90 patients with AML were included. AML patients were stratified into three groups: [1] those with a WBC count below 50 × 109/L (n = 33), [2] those with a WBC count 50–100 × 109/L (n = 29) and [3] those with a WBC above 100 × 109/L (n = 28). Complete blood cell count, immunophenotyping and PCR of the exon 12 of NPM1 gene followed by sequencing analysis were done. NPM1 mutations were detected in 20/90 (22.2%) of patients with AML involving exon 12 of NPM1 gene and showed significant correlations with some hematologic characteristics. Contrary to expectation, no statistically significant difference was found in the WBC counts according to the NPM1 gene the mutation status among the studied groups. Also, there were no significant differences in the WHO classification categories according to the HL grading score. The difference in the frequency of NPM1 mutation in Asian compared to Western patients might be attributed to biology and aetiology variation of the disease in different population. The current two cut off values defining the HL are molecularly unreliable in spite of being clinically defined.

Prospective Evaluation of Allogeneic HSCT at the First Remission for Younger Adults with FLT3-ITD Positive Acute Myeloid Leukemia: The JALSG AML209-FLT3-SCT Study

Prospective Evaluation of Allogeneic HSCT at the First Remission for Younger Adults with FLT3-ITD Positive Acute Myeloid Leukemia: The JALSG AML209-FLT3-SCT Study

Older Patients with NPM1 Mutated AML Have Distinctive Genomic Mutation Landscape Associated with Enrichment in Immunosuppressive Gene Signature

Background: Mutations in the nucleophosmin (NPM1) gene are associated with better responses to chemotherapy and improved survival among acute myeloid leukemia (AML) patients. However, older AML patients (≥ 60 years old) with NPM1 mutation have worse survival outcomes than younger patients (&amp;lt;60 years old). This may be attributed to more adverse biologic features (frequent complex karyotype, FLT3 mutations) in addition to lower odds to receive intensive curative chemotherapy due to co-morbidities. We sought to compare the outcomes of older NPM1 mutated AML patients with younger NPM1 mutated patients after exclusions of patients with adverse-risk per ELN 2017 criteria. We also compared their genomic mutation profile and gene expression utilizing the Beat AML dataset. Methods: We queried the Beat AML dataset, supported in part by the Leukemia &amp; Lymphoma Society and the OHSU Knight Cancer Institute, for pts with NPM1 gene mutations who did not have adverse-risk ELN 2017 (poor cytogenetic profile or mutations in FLT3, TP53 or ASXL1). Descriptive statistics described baseline characteristics and responses. Kaplan-Meier with log-rank test was used for survival analysis. DNA mutation data were obtained from the exome sequencing and analyzed using the beat AML data viewer (Vizome). RNA exome sequencing data were downloaded. Differential expression of raw count RNA-Seq and gene set enrichment was done using R via limma and ClusterProfiler packages. Results: Among 562 unique patients in the Beat AML umbrella trial, there were 81 patients with newly diagnosed NPM1 mutated AML after exclusion of patients with ELN 2017 adverse-risk category. Among these patients there was 49 older patients (≥ 60 years old) and 32 younger patients (&amp;lt;60 years old). 39 (77.6%) in the older group received intensive induction chemotherapy and 30 patients (93.7%) in the younger group. 29 (59.1%) patients achieved complete morphologic responses in the older patient group compared to 28 (84.4%) in the younger patient group (OR 0.2, P=0.009). Median overall survival in the older patient group was 20.1 months compared to 25.4 months in the younger group (HR 0.52, P=0.08). Exome sequencing data were available for 43 and 30 patients from the older and younger group respectively. There was a median of 6.5 (2-20) and 7 (2-19) mutations in the older and younger group respectively (P=0.78). After exclusion of the benign mutations and variant of unknown significance, the median number of mutations was 4 in both group (P=0.28). Both groups shared only 24 (3.9%) of the gene mutations while there were 334 unique gene mutations in the older group and 262 in the younger group. Most common gene mutations were DNMT3a, TET2, NRAS, WT1, and PTPN11 with frequencies are shown (Figure 1). RNA sequencing data was available for 26 patients from the older group and 18 patients from the younger group. We explored the gene expression profile of the top 1000 differentially expressed genes in both groups after adjustment. There was distinctive clustering of the gene expression profile between the two groups (Figure 2). Gene set enrichment analysis identified multiple immune-related pathways among the highly enriched gene sets in both groups but with different functions in the two groups. There was significant gene set enrichment in the TGFβ signaling in the older patient group which is associated with immune suppression and microenvironment modulation. While the younger group showed significant enrichment in the TNFa, IL17, PI3K-AKT signaling which are associated with inflammation. Conclusion: Older AML patients with NPM1 mutations, and no adverse risk features, had lower rate of complete responses and a trend towards a worse survival compared to younger patients. Whole exome sequencing did not show increased mutational burden. However, 96% of the mutated genes were different between the two groups as were the gene expression profiles. Gene set enrichment analysis showed contrasting enriched immune-related pathways between both groups. The immunosuppressive TGFβ signaling gene set were significantly enriched in the older group while the inflammatory TNFa, IL17, PI3K-AKT signaling gene sets were significantly enriched in the younger group. Older AML patient with NPM1 mutations have distinctive genomic landscape compared to the younger patient which may explain in part the worse clinical outcomes in the absence of other adverse risk features. Disclosures Lin: Jazz Pharmaceuticals: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees.

Mutational Profiling Predicts Clinical Outcomes to Azacytidine and Low Dose Cytarabine Plus Fludarabine (FLUGA) in Elderly Acute Myeloid Leukemia Patients Enrolled in the Pethema Phase III Flugaza Trial

Mutational Profiling Predicts Clinical Outcomes to Azacytidine and Low Dose Cytarabine Plus Fludarabine (FLUGA) in Elderly Acute Myeloid Leukemia Patients Enrolled in the Pethema Phase III Flugaza Trial

NPM1 mutations Using Deep Amplicon Sequencing and Broad Next Generation Sequencing at the Time of Complete Remission Is Informative to Predicting Risk of Relapse Following Intensive Chemotherapy

Introduction: NPM1 gene mutations are a common molecular aberration in acute myeloid leukemia (AML). In the absence of concurrent high FLT3-ITD ratio mutations (&amp;gt;0.5), NPM1 mutations typically associate with higher complete remission (CR) rates following intensive induction chemotherapy. NPM1 mutations have been shown to be stable markers of persistent disease or impending relapse during CR or complete remission with incomplete count recovery (CRi). Given the clinical implications that persistent NPM1 mutations can have during CR/CRi, we used Deep Amplicon sequencing on CR/CRi bone marrow (BM) samples collected from adult de novoNPM1-mutated AML patients to determine the ability of NPM1 mutations at both a high and lower sensitivity next generation sequencing methods and also the presence of additional clonal abnormalities on relapse risk. Methods: We performed targeted next generation sequencing (NGS) analysis in addition to NPM1 Deep Amplicon sequencing on paired BM or blood samples collected from 38 newly diagnosed NPM1-mutated AML patients during CR/CRi after successful induction (1-2 courses of 7 + 3) and, if available, at relapse. NPM1 mutated NGS libraries were prepared using a KAPA HyperPlus Kit (Roche, Pleasanton, CA) and xGen Lockdown Probes (IDT, Coralville, IA). Libraries were sequenced using the Illumina HiSeq 4000 (Illumina, San Diego, CA). GATK's MuTect2 was used to perform variant calling. Variant allele frequency (VAF) cut-off for the NGS panel was 0.05 (5%) with the exception of hotspot variants in IDH1 (R132) and IDH2 (R140) where variants detected to a level of 0.01 (1%) were included. The VAF cut off used for NPM1 Deep Amplicon sequencing was 0.00012 (0.012%). Results: Targeted NGS analysis and NPM1 Deep Amplicon sequencing had exceptional concordance at the level of detection of VAF= 0.05 (Figure 1). Of 38 patients, 23 patients had undetectable NPM1 mutations as analyzed through NPM1 Deep Amplicon sequencing of whom 9 (38.1%) relapsed. In contrast, 15 patients were positive by NPM1 Deep Amplicon sequencing and 9 (60%) relapsed. Only 4 patients had detectable persistent NPM1 mutations after induction according to both detection techniques and two of these relapsed. We next examined the potential impact of clearing both NPM1 mutation and co-occurring mutations together on relapses (Figure 2). A total of 15 patients cleared all of their clonal abnormalities and 5 (27%) relapsed. In contrast, of the 23 patients who did not clear the NPM1 mutation and/or another co-occurring mutation at remission, 14 (61%) have relapsed. Eleven of the relapsed patients had relapse samples available of whom all had persistent NPM1 mutation at this time. Paired CR/CRi and relapsed samples showed acquisition or recurrence of several other mutations, most notably FLT3-ITD, IDH1, and IDH2 which are all targetable with small molecule therapeutics. Conclusions: The use of Deep Amplicon sequencing to identify NPM1 mutations at a lower detection threshold compared to standard NGS techniques was more sensitive, but did not appear to fully inform relapse rates in NPM1-mutated AML patients after receipt of induction therapy. The appearance of other AML-associated mutations, identified together with NPM1 at time of remission, was more frequent among patients relapsing. These pilot data provide support for concurrent assessment of Deep Amplicon sequencing together with a broad standard NGS AML mutational assay to further enhance risk stratification of NPM1-mutated patients. Additionally, while NPM1 clones are present in all patients examined at the time of relapse, persistence or development of targetable clones justifies repeat broad NGS sequencing at this time. Figure Disclosures Bhatnagar: Novartis and Astellas: Consultancy, Honoraria; Cell Therapeutics, Inc.: Other: Research support; Karyopharm Therapeutics: Other: Research support. Mims:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees. Behbehani:Fluidigm corporation: Other: Travel funding. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents &amp; Royalties: OSU-2S.

Value of molecular genetics combined with bone marrow imaging detection in the prognosis judgement of acute myeloid leukemia with normal karyotype

Objective To investigate the value of NPM1 and FLT3 gene mutation combined with bone marrow imaging detection in the prognosis judgement of initial treatment cytogenetically normal acute myeloid leukemia (CN-AML). Methods The clinical data of 100 patients (non-M3 type) with primary and initial treatment CN-AML from January 2010 to January 2014 in the Peace Hospital Affiliated of Changzhi Medical College were retrospectively analyzed. All patients were enrolled in the bone marrow imaging examination on the end day of induction treatment or the first day after the end of induction treatment (T time point). Univariate and multivariate prognostic analyses were performed on AML patients according to FLT3 and NPM1 gene status,bone marrow juvenile cell ratio at T time point. Results A total of 100 patients included 36 cases with FLT3 gene mutation and 44 cases with NPM1 gene mutation. The complete remission (CR) rate of CN-AML patients was 13.9% (5/36) and 71.9% (46/64), respectively (P 0.05). The CR rate, 2-year RFS rate and 2-year OS rate in NPM1+ FLT3- group were better than those in NPM1- FLT3-, NPM1- FLT3+ and NPM1+ FLT3+ groups; NPM1- FLT3+ and NPM1+ FLT3+ groups had the worst prognosis, and there were no statistical differences in the CR rate, RFS and OS time between the two groups (all P > 0.05). The prognosis of the patients in bone marrow juvenile cell ratio 0.05). The medium prognosis group+ the ratio of bone marrow juvenile cells at T time point ≥0.05 group, the poor prognosis group+ the ratio of bone marrow juvenile cells at T time point < 0.05 group had the equivalent prognosis, and the average prognosis was moderate; the poor prognosis group+ the ratio of bone marrow juvenile cells at T time point ≥ 0.05 group had the worst prognosis. According to Cox multivariate regression analysis, FLT3 gene mutation and the ratio of bone marrow juvenile cells at T time point were independent influencing factors for RFS and OS in CN-AML patients (all P < 0.05). NPM1 was an independent prognosis factor affecting RFS and OS of FLT3- patients (all P < 0.05). Conclusions After induction chemotherapy, the responsiveness and sensitivity of AML patients to chemotherapy regimen can be assessed early and objectively according to molecular genetics and the ratio of bone marrow juvenile cells at T time point, which has a certain value in the prognosis judgement. Key words: Leukemia, myeloid, acute; Molecular biology; Bone marrow examination; Gene, NPM1; Gene, FLT3; Prognosis

Transcriptome analysis offers a comprehensive illustration of the genetic background of pediatric acute myeloid leukemia

AbstractRecent advances in the genetic understanding of acute myeloid leukemia (AML) have improved clinical outcomes in pediatric patients. However, ∼40% of patients with pediatric AML relapse, resulting in a relatively low overall survival rate of ∼70%. The objective of this study was to reveal the comprehensive genetic background of pediatric AML. We performed transcriptome analysis (RNA sequencing [RNA-seq]) in 139 of the 369 patients with de novo pediatric AML who were enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 trial and investigated correlations between genetic aberrations and clinical information. Using RNA-seq, we identified 54 in-frame gene fusions and 1 RUNX1 out-of-frame fusion in 53 of 139 patients. Moreover, we found at least 258 gene fusions in 369 patients (70%) through reverse transcription polymerase chain reaction and RNA-seq. Five gene rearrangements were newly identified, namely, NPM1-CCDC28A, TRIP12-NPM1, MLLT10-DNAJC1, TBL1XR1-RARB, and RUNX1-FNBP1. In addition, we found rare gene rearrangements, namely, MYB-GATA1, NPM1-MLF1, ETV6-NCOA2, ETV6-MECOM, ETV6-CTNNB1, RUNX1-PRDM16, RUNX1-CBFA2T2, and RUNX1-CBFA2T3. Among the remaining 111 patients, KMT2A-PTD, biallelic CEBPA, and NPM1 gene mutations were found in 11, 23, and 17 patients, respectively. These mutations were completely mutually exclusive with any gene fusions. RNA-seq unmasked the complexity of gene rearrangements and mutations in pediatric AML. We identified potentially disease-causing alterations in nearly all patients with AML, including novel gene fusions. Our results indicated that a subset of patients with pediatric AML represent a distinct entity that may be discriminated from their adult counterparts. Based on these results, risk stratification should be reconsidered.

Clinical Features and Prognosis of 188 Patients with Acute Myeloid Leukemia-M2

To summarize the clinical characteristics of patients with acute myeloid leukemia-type M2 (AML-M2) and analyze the factors affecting the prognosis. One hundred eighty-eight AML-M2 patients were retrospectively analyzed for the following parameters including peripheral blood, immune phenotypes, fusion genes and cytogenetics to explore their significance for the overall survival (OS) and progression-free survival (PFS). The prognostic factors were also analyzed. Among 188 patients with AML-M2, the chromosomal abnormality with t (8;21), normal chromosome and other abnormalities accounted for 37% (70/188), 41% (77/188) and 22% (41/188), respectively. For the immunopheno typing of M2 patients, the hematopoietic progenitor cell differentiation antigen CD117 (96.1%) were mainly expressed, CD34 (81.6%) and HLA-DR (55.9%), and myeloid-associated antigen of CD13 (90.5%) and CD33 (89.4%) were also highly expressed. There were lymphoid-associated antigens expressed in some patients, among which the positive expression rate of CD19 was highest (29.6%), and the next was CD7 (28.5%). The most common accompanied mutations was FLT3 mutation (30.2%). The univariate analysis showed that the patients at age＜50 years old, without extramedullary infiltration, with positive expression of CD19, NPM-1 (-), CEBPA double mutation(+), and HSCT were significant superior in OS and PFS (P＜0.05); the multivariate analysis showed that the patient at age＜50 years old, without extramedullary infiltration, with positive expression of CD19 and CEBPA double mutation (+) were significant superior in OS and PFS (P＜0.05). The analysis indicated that the Karytypes affected only OS (P＜0.05), while the NPM-1 gene mutation positive affected only PFS (P＜0.05). The univarate analysis of factors affecting the survival in 70 AML-M2 patients with t (8;21) abnormatity showed that the C-KIT gene mutation was a poor factor for OS and PFS. The clinical characteristics are different between M2 patients with different karyotype, and prognostic analysis shows that the karytypes have an impact on overall survival; age, extramedullary infiltration, CD19 expression and CEBPA double mutation are also the main factors impacting the prognosis of patients.

The Value of FLT3, NPM1 and DNMT3A Gene Mutation Analysis in Acute Myeloid Leukemia Diagnosis

The Value of FLT3, NPM1 and DNMT3A Gene Mutation Analysis in Acute Myeloid Leukemia Diagnosis

PB1764 THE ANALYSIS OF GENE MUTATIONS IN PATIENTS WITH ACUTE MYELOID LEUKAEMIA

Background:Most AML patients present different cytogenetic and molecular defects associated with certain biologic and clinical features of the disease. Approximately 40–50 % of de novo AML patients demonstrate chromosomal aberrations. About half of the cases of AML are patients with normal karyotype from the subgroup with intermediate prognosis. However, phenotypically this group is heterogeneous and can be divided into prognostically different subgroups. Knowledge of genetic changes in AML patients allows to fully represent the development of the disease and assess the prognostic features of a particular variant of AML.Aims:To study the role of gene mutations in the diagnosis and prediction of AML.Methods:111 patients with AML (median age 53.2 years) were examined. DNA samples from blood and bone marrow were isolated by standard SDS‐method. Mutations FLT‐ITD and NPM1 were analyzed by PCR with specific primers. The FLT3 D835 mutation was analyzed using the RFLP‐PCR method with EcoRV restriction. CEBPA and p53 gene mutations were detected by SSCP‐TCR within the entire coding sequence of the gene.Results:In the study group, the mutation rate of FLT3‐ITD was 21.6 %, mutations of FLT3‐D835 ‐ 8%, mutations of NPM1 ‐ 21.6 %, mutations of CEBPA ‐ 11.3 %, mutations of p53 gene‐10.3 %. Statistically significantly more common mutations was evaluated in patients with a normal karyotype (p = 0.003) and in the group of intermediate prognosis (p = 0.001) compared with the group of AML with a poor karyotype. Depending on the AML variant, there were no differences in the mutation frequency of FLT3‐ITD (M0 11%, M1 17%, M2 24%, M3 32%, M4 17.6%). Mutations of FLT3 D835 were observed more often in M3 (16%) and M0 (11%) variants. Patients with M1 and M4 variants had no FLT3‐D835 mutation at all. NPM1 gene mutations were more common in M1 (28.6%) and M4 (24.5%) variants, in M2 and M3 17% and 16% respectively. Patients with M0 variant had no NPM1 gene mutation. When dividing patients with AML by age, there was no difference depending on the mutation status. Median age of patients with mutations in FLT3 gene was 50.6 (18…80) years, patients with NPM1 mutations – 55.9 (20…80) years. Patients with FLT3‐ITD and FLT3 ‐ D835 mutations had a higher number of leukocytes in the blood than in the group with NPM1 mutation (p = 0.022) and in the group without mutations (p = 0.003). Patients with FLT3‐ITD mutations had a higher content of blast cells in the bone marrow than patients without mutation (p = 0.038). The frequency of combined mutations in AML patients was 11.7%, of which FLT3‐ITD+NPM1‐8.1%, FLT3 D835+ NPM1 ‐ 0.9% and FLT3‐ITD + FLT3 D835 – 2.7%. Depending on the mutation status, the lowest overall survival was in patients with mutations FLT3‐ITD and FLT3‐ITD+FLT3‐D835. In the group of patients with a combination of mutation FLT3‐ITD+NPM1 overall survival did not differ from the group of patients without mutations (p = 0.112).Summary/Conclusion:The study of mutational status of genes FLT3 and NPM1 improves diagnosis of AML patients and allows to expand the group of prognostic markers. Flt3‐ITD mutation is associated with increased levels of leukocytes in peripheral blood and blasts in the bone marrow, as well as a negative impact on patient survival.

PF544 THE DIFFERENT MUTATIONAL LANDSCAPES OF A PANEL GENES BY NEXT GENERATION SEQUENCING IN 967 CASES OF MDS AND AML

Background: Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are myeloid disease with dysplasia characterized by multiple gene mutations in most cases. With advances in Next-generation sequencing techniques, the molecular landscapes in MDS or AML have been reported with further understanding the disease pathogenesis. As well known, MDS may end up in evolving into AML in most cases, especially the subtypes of MDS with excessive blasts. The driver mutation in MDS developing to AML is still open to study. The comparison of mutational landscapes between MDS and AML will provide evidence to better understand the underlying molecular mechanisms of the myeloid dysplasia evolution. Aims: To analyze the different mutational status of 22 genes commonly observed in MDS and AML. Methods: The bone marrow samples were collected from patients with AML (749 cases) and MDS (218 cases) with written consent from June 2017 to January 2019. Next generation sequencing of a panel of 22 genes was used to evaluate the mutations in MDS/AML patients. Results: 719/ 967(74.4%) cases with MDS/AML were detected at least one gene mutation. There were 274 gene mutations in 142/ 218 (65.1%) cases with MDS and 1224 gene mutations in 577/749(77.0%) cases with AML. Both the median mutations and the frequency of gene mutation between MDS and AML were significantly difference (p < 0.05). Compared to MDS, the mutation rate of CEBPA (11.10% vs 1.40%, p < 0.01), NRAS (14.00% vs 6.40%, p < 0.01), KIT (7.30% vs 0.00%,p < 0.01), NPM1 (17.80% vs 5.00%, p < 0.01), FLT3-ITD (15.40% vs 0.90%, p < 0.01), DNMT3A (10.90% vs 4.10%, p < 0.01), IDH2 (9.5% vs 2.8%, p < 0.01), and IDH1 (6.00% vs 1.40%, p < 0.01) genes were significantly higher in AML. While the mutation rate of ASXL1 (5.30% vs 14.7%, p < 0.01), U2AF1 (5.90% vs 22.00%, p < 0.01), RUNX1 (6.30% vs 10.60%, p < 0.05), SRSF2 (2.30% vs 6.00%, p < 0.05), EZH2 (0.50% vs 3.20%, p < 0.01), SETBP1 (0.70% vs 5.00%, p < 0.01), SF3B1 (2.50% vs 7.80%, p < 0.01), and CBL (0.80% vs 3.20%, p < 0.05) genes were significantly higher in MDS. There were no significant difference of the incidence in TET2, FLT3-TKD, JAK2, ETV6, TP53, PHF6 and ZRSR2 gene mutations. Considering the gene mutation increasing with age, the cases were divided into the young (age < 60 years old) and the old (age≥60 years old). In the young cases, the gene mutation profile was similar to the cohort with incidences of CEBPA, NRAS, KIT, NPM1, FLT3-ITD, DNMT3A, IDH1, plus TET2 gene mutations were significantly higher in AML (p < 0.05). The incidences of ASXL1, U2AF1, TP53, EZH2, SETBP1, and CBL gene mutations were significantly higher in MDS (p < 0.05), and the incidences of FLT3-TKD, JAK2, RUNX1, IDH2, ETV6, PHF6, SRSF2, SF3B1 and ZRSR2 gene mutations were not significantly different between AML and MDS cases (p>0.05). The old cohort showed a different gene mutation profile as compared to the young cohort with the incidences of CEBPA, NPM1, FLT3-ITD, and IDH2 gene mutations were significantly higher in AML (p < 0.01), and the incidences of U2AF1 and SF3B1 gene mutations were significantly higher in MDS. There were no significant differences of all the other gene mutations between AML and MDS cases in the old cohort. Summary/Conclusion: There are typical gene mutation patterns in MDS and AML cases. Some genes mutate in MDS and AML cases without differences and others mutate mainly in AML or MDS. The roles and significances of these gene mutation profiles warranted further investigations to understand the pathogenesis underlying MDS/AML.

PB1738 NUCLEOPHOSMIN‐1 GENE MUTATION DETECTION: COMPARATIVE IMMUNO‐STAINING STUDY FOR BONE MARROW (BM) BIOPSY VERSUS BM ASPIRATE &amp; BM CLOT IN DE NOVO ADULT ACUTE MYELOID LEUKEMIA PATIENTS AMONG EGYPTIANS

Background:NPM1 gene mutation is a well‐known prognostic marker in AML, probably related to its cytoplasmic localization. NPM1 gene mutation detection could be done by either molecular genetics or its protein product by immunohistochemical staining of bone marrow (BM) biopsy.Aims:This study aimed to compare the detection rate of the immuno‐staining technique for mutated nucleophosmin‐1 gene when applied on the standard BM biopsy versus BM aspirate or BM clot biopsy in adult de novo patients of acute myeloid leukemia.Methods:Forty adult patients (18‐60 years) with newly diagnosed AML were enrolled into this study. Detection of nucleophosmin localization either nucleolar or cytoplasmic was performed using monoclonal mouse anti‐human nucleophosmin (clone 376) by conventional immunohistochemical technique on BM biopsy, BM aspirate &amp; BM clot biopsy.Results:The study included 40 AML patients, 23 males, 17 females. The complete remission (CR) rate was 70%. Median overall survival (OS) was 2.5 month (0.1‐40 months). Median disease free survival was 6 month (0.2‐39). NPM1 was detected in 11/40 patients by conventional immunohistochemical technique on BM biopsy, 11/39 patients by BMA technique, and 9/29 by BM clot technique. The BMA technique had Sensitivity of 100%, Specificity of 78.6%, positive predictive value of 64.7%, negative predictive value of 100%, and diagnostic accuracy of 84.6%. The BM clot technique had sensitivity of 77.8%, specificity of 90.0%, and positive predictive value of 77.8%, negative predictive of 90.0%, and diagnostic accuracy 86.2%.Summary/Conclusion:The BMA technique had good sensitivity and so could be used as a cheap screening method even in patients with profound thrombocytopenia. BM clot technique had good specificity. Both BMA and BM clot techniques could be a reasonable cheap option if combined together in cases in which BM biopsy could not be done.image