scholarly journals Loss of Heterozygosity of FLT3-ITD Is Common in Acute Myeloid Leukemia and May be a More Consistent Prognostic Marker Than FLT3-ITD Allele Frequency

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1437-1437
Author(s):  
Eric A. Severson ◽  
Ethan S. Sokol ◽  
Russell Madison ◽  
Daniel Duncan ◽  
Amanda Hemmerich ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Allele Frequency ◽  
Point Mutation ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Wild Type ◽  
Tumor Purity ◽  
Equity Ownership ◽  
Acute Myeloid ◽  
Control Samples

BACKGROUND: FLT3 alterations in Acute Myeloid Leukemia (AML) occur as either point mutations in the tyrosine kinase domain (TKD) or internal tandem duplications (ITD), both of which result in constitutive activation of FLT3; however, only FLT3-ITD has prognostic value. Per NCCN guidelines, when NPM and FLT3-ITD alterations co-occur, an allele frequency ≥ 0.5 for FLT3-ITD (FLT3-ITDhigh) confers an intermediate prognosis, while allele frequencies < 0.5 (FLT3-ITDlow) confers a favorable prognosis. For allele frequency to be ≥ 0.5, loss of heterozygosity (LOH) or copy number gains at the FLT3 locus are required. In this study, we examined a large cohort of FLT3 mutated AML samples to study LOH at the FLT3 locus. DESIGN: During routine clinical care, 2129 AML samples were evaluated by comprehensive genomic profiling (CGP) for 406 genes via DNAseq for all classes of genomic alterations and 265 genes via RNAseq for rearrangements, using a hybrid-capture next generation sequencing assay (FoundationOne®Heme). Of these samples, 1379 met analytic specifications required for LOH analysis. LOH analysis was performed by first generating copy number models from exon and SNP log ratio and minor allele fractions (maf). LOH was then determined using the modeled copy number, maf, and tumor purity. Samples with low aneuploidy were reviewed manually. RESULTS: The median age of the overall cohort was 60 y (range <1y-88y) with 43.4% females and 56.6% males. Of 1379 samples, 265 (19%) had at least one functional alteration in FLT3. There were 171 (12.4%) samples with FLT3-ITD alteration(s), 115 (8.3%) samples with FLT3 TKD alteration(s), and 21 (1.5%) samples with both FLT3-ITD and TKD alterations. LOH analysis was performed for 236 AML samples with a FLT3 alteration and 224 randomly selected wild-type FLT3 AML samples as controls. Samples with a FLT3 alteration had a significantly higher rate of LOH compared to the control samples (34/236 (14.4%) versus 2/224 (0.9%), p = 9.0x10-9). Samples with a FLT3-ITD were under significantly greater LOH than samples with a point mutation (33/171 (19.3%) versus 4/115 (3.5%), p = 2.8x10-5). Samples with only a point mutation rarely had LOH (1/94, 1.0%), not significantly different than FLT3 wild-type controls. Copy number analysis revealed that FLT3-altered samples had a rate of FLT3 copy number gains not significantly different than control samples (10/236 (4.2%) versus 7/224 (3.1%)). The allele frequency was significantly higher for FLT3-ITD alterations compared to FLT3-TKD alterations (median 0.11 versus 0.24, p < 0.001), and FLT3-ITD alterations under LOH had a higher allele frequency than those not under LOH (median 0.35 versus 0.24, p< 0.01). Only 1/115 FLT3-TKD alterations had an allele frequency > 0.5 versus 7/171 of the FLT3-ITD alterations. Five (of 7) FLT3-ITDhigh samples were under LOH and the remaining 2 samples had FLT3 copy number gains. FLT3-ITD mutated AML has co-occurring genomic alterations of clinical significance; however, with this sample size there were no statistically significant differences in the frequencies between samples with and without LOH at the FLT3 locus (NPM1: 64% vs 43%, DNMT3A: 48% vs 37%, WT1: 36% vs 25%, RUNX1: 12% vs 17%, NRAS: 3% vs 16%). CONCLUSIONS: In AML, the FLT3 locus has increased LOH when a FLT3-ITD is present, compared to FLT3 wild-type controls and samples with FLT3-TKD alterations. Copy number alterations in FLT3 are not different in FLT3-altered vs FLT3 wild type AML. Allele frequencies were higher for FLT3-ITDs compared to FLT3-TKDs and were highest for FLT3-ITDs under LOH. An emerging negative prognostic indicator in AML is FLT3-ITDhigh. Determination of FLT3-ITDhigh status requires high tumor purity, copy number gains, and/or LOH. The requirement of high tumor purity makes FLT3-ITDhigh status dependent on factors other than tumor biology, such as tumor sampling. In our dataset, 33 (19%) FLT3-ITD samples were under LOH but only 5 (4%) were FLT3-ITDhigh. LOH of FLT3-ITDs has also been associated with worse prognosis in AML and further studies are warranted to determine if allele frequency or absence of wild-type FLT3 drives these prognostic correlations. FLT3-ITD LOH is more common than FLT3-ITDhigh and could provide more accurate, sample independent risk stratification for patients with FLT3-ITD+ AML. Disclosures Severson: F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Sokol:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Madison:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Duncan:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Hemmerich:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Edgerly:Foundation Medicine, Inc: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Huang:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Britt:Foundation Medicine, Inc: Employment. Vergilio:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Elvin:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Reddy:Foundation Medicine, Inc: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Sathyan:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Alexander:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment. Ross:Foundation Medicine, Inc.: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ali:Foundation Medicine, Inc: Employment; F. Hoffman La Roche, Ltd.: Equity Ownership. Ramkissoon:F. Hoffman La Roche, Ltd.: Equity Ownership; Foundation Medicine, Inc.: Employment.

scholarly journals Changes of the Mutational Landscape in Relapsed Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 599-599
Author(s):  
Franck Rapaport ◽  
Marc Robert de Massy ◽  
Adil al Hinai ◽  
Mathijs A. Sanders ◽  
Todd Hricik ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Allele Frequency ◽  
Combination Chemotherapy ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Variant Allele ◽  
Chemotherapy Treatment ◽  
Variant Allele Frequency ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. Unfortunately, a significant proportion of patients relapse after responding to initial treatment reflecting our poor understanding of the mechanisms mediating therapy resistance and relapse. We hypothesized that understanding the evolution of the mutational landscape between diagnosis and relapse is essential in order to identify mutational markers associated with sensitivity or resistance to treatment. To address this hypothesis we assembled a cohort of 53 clinically annotated, paired AML patient samples (diagnosis, relapse and patient-matched germline samples; mean age = 52 years). All patients achieved clinical remission after treatment with combination chemotherapy (cytarabine arabinoside and an anthracycline) during induction phase followed by consolidation chemotherapy treatment with or without a stem cell transplantation in first remission. Serial samples were collected at the time of initial diagnosis and within three months of relapse (mean time to relapse 455 days). We performed whole-exome and targeted capture followed by high-throughput sequencing. We aligned samples with BWA, recalibrated them with The Genome Analysis Toolkit (GATK) and then compiled integrated calls from substitution and indel callers (Mutect, Scalpel, Strelka, Varscan and Somatic Sniper). We performed several layers of post-processing filtering on these calls, including removing non-oncogenic mutations and previously documented non-somatic variants, and correcting for the variant allele fraction of indel calls. We filtered out the variants that were found to occur in non-copy number neutral re-arrangements using the clinically determined cytogenetic data. Furthermore, we assessed for copy number events, including loss of heterozygosity events, and for the presence and the variant allele frequency of the FLT3-ITD in our samples. We observed a median of 4.5 and 5 mutations per patient at diagnosis and relapse, respectively, with 3.5 mutations being shared by paired diagnosis and relapse samples. When limiting our analysis to genes previously shown to contribute to leukemogenesis, we found a median of 1.5 and 2 mutations per patient at diagnosis and relapse, with 1 mutation being shared. FLT3, DNMT3A, IDH2, NRAS, RUNX1 and TET2 were among the most commonly mutated genes, with a detected presence rate of 28%, 25%, 19%, 19%, 11% and 11%, respectively, in the diagnosis samples and 39%, 23%, 19%, 4%, 13% and 11% in the relapse samples. We identified significant variation in the variant allele frequency (VAF) for several of the mutations related to these genes and others, denoting variations in the cellular prevalence of the related clones after adjustment for tumor content using the mutations with the highest VAF to delineate clonal architecture. Specifically, we observed that DNMT3A, IDH2, TET2 variants are most commonly present in the bulk AML clone, and persist after treatment. WT1, GATA2 and FLT3mutations are predicted to confer relative resistance to standard combination chemotherapy treatment based on their increased VAF at relapse, whereas KRAS and NRAS subclone(s) are more sensitive to chemotherapy since their VAFs decrease following multiagent chemotherapy. Fifteen patients presented new events in leukemogenesis-related genes at relapse. Overall, our results support a model of AML as a disease with a complex mutational hierarchy and clonal architecture and provide further insight into how these change in response to standard induction therapy. Our data suggests that future efforts to develop targeted therapies with maximal clinical benefit in combination with standard induction treatments should be placed on mutated genes identified to be more strongly associated with disease relapse. Authors contributed equally: F. Rapaport and M.R. De Massy Authors contributed equally: A. al Hinai and M.A. Sanders Disclosures Guzman: Cellectis: Research Funding. Roboz:Cellectis: Research Funding; Agios, Amgen, Amphivena, Astex, AstraZeneca, Boehringer Ingelheim, Celator, Celgene, Genoptix, Janssen, Juno, MEI Pharma, MedImmune, Novartis, Onconova, Pfizer, Roche/Genentech, Sunesis, Teva: Consultancy. Melnick:Janssen: Research Funding. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

scholarly journals Phase I/II Study of Combination Therapy With Sorafenib, Idarubicin, and Cytarabine in Younger Patients With Acute Myeloid Leukemia

2010 ◽  
Vol 28 (11) ◽  
pp. 1856-1862 ◽  
Author(s):  
Farhad Ravandi ◽  
Jorge E. Cortes ◽  
Daniel Jones ◽  
Stefan Faderl ◽  
Guillermo Garcia-Manero ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Complete Remission ◽  
Phase I ◽  
Myeloid Leukemia ◽  
Wild Type ◽  
Platelet Recovery ◽  
Probability Of Survival ◽  
Target Effect ◽  
Acute Myeloid

Purpose To determine the efficacy and toxicity of the combination of sorafenib, cytarabine, and idarubicin in patients with acute myeloid leukemia (AML) younger than age 65 years. Patients and Methods In the phase I part of the study, 10 patients with relapsed AML were treated with escalating doses of sorafenib with chemotherapy to establish the feasibility of the combination. We then treated 51 patients (median age, 53 years; range, 18 to 65 years) who had previously untreated AML with cytarabine at 1.5 g/m2 by continuous intravenous (IV) infusion daily for 4 days (3 days if > 60 years of age), idarubicin at 12 mg/m2 IV daily for 3 days, and sorafenib at 400 mg orally twice daily for 7 days. Results Overall, 38 (75%) patients have achieved a complete remission (CR), including 14 (93%) of 15 patients with mutated FMS-like tyrosine kinase-3 (FLT3; the 15th patient had complete remission with incomplete platelet recovery [CRp]) and 24 (66%) of 36 patients with FLT3 wild-type (WT) disease (three additional FLT3-WT patients had CRp). FLT3-mutated patients were more likely to achieve a CR than FLT3-WT patients (P = .033). With a median follow-up of 54 weeks (range, 8 to 87 weeks), the probability of survival at 1 year is 74%. Among the FLT3-mutated patients, 10 have relapsed and five remain in CR with a median follow-up of 62 weeks (range, 10 to 76 weeks). Plasma inhibitory assay demonstrated an on-target effect on FLT3 kinase activity. Conclusion Sorafenib can be safely combined with chemotherapy, produces a high CR rate in FLT3-mutated patients, and inhibits FLT3 signaling.

scholarly journals High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1517-1524 ◽  
Author(s):  
Marjan J. T. Veuger ◽  
M. Willy Honders ◽  
Jim E. Landegent ◽  
Roel Willemze ◽  
Renée M. Y. Barge
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Messenger Rna ◽  
Myeloid Leukemia ◽  
Deoxycytidine Kinase ◽  
Wild Type ◽  
Healthy Donors ◽  
Alternatively Spliced ◽  
Acute Myeloid

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

scholarly journals TP53 Action and the Consequences of TP53 Mutation in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-13-SCI-13
Author(s):  
Scott W. Lowe
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Tp53 Mutation ◽  
P53 Mutation ◽  
Tp53 Mutations ◽  
Genomic Analyses ◽  
Equity Ownership ◽  
Myeloid Neoplasia ◽  
The Impact ◽  
Acute Myeloid

p53 action and the consequences of p53 mutation in acute myeloid leukemia TP53 mutations are common in treatment associated myeloid neoplasia (tMN) and complex karyotype acute myeloid leukemia (CK-AML), where they are associated with chemoresistance and one of the worst prognoses of any leukemia genotype. To understand the impact of TP53 mutations on AML biology, we are performing arge scale genomic analyses of p53 mutant AML and have produced a series of animal models that appear to faithfully reflect molecular and biological features of the human disease. We have gone on to explore the biology of particular TP53 mutational configurations drive AML initiation and maintenance, and to identify and understanding the events that cooperate with p53 mutations during leukemogenesis. Disclosures Lowe: Blueprint Medicines: Consultancy, Equity Ownership; ORIC pharmaceuticals: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership.

scholarly journals Epigenetic Activation of the pH Regulator MCT4 in Acute Myeloid Leukemia Exploits a Fundamental Metabolic Process of Enhancing Cell Growth through Proton Shifting

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3765-3765
Author(s):  
Cheuk-Him Man ◽  
David T. Scadden ◽  
Francois Mercier ◽  
Nian Liu ◽  
Wentao Dong ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Growth ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Monocarboxylate Transporter ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Acute Myeloid

Acute myeloid leukemia (AML) cells exhibit metabolic alterations that may provide therapeutic targets not necessarily evident in the cancer cell genome. Among the metabolic features we noted in AML compared with normal hematopoietic stem and progenitors (HSPC) was a strikingly consistent alkaline intracellular pH (pHi). Among candidate proton regulators, monocarboxylate transporter 4 (MCT4) mRNA and protein were differentially increased in multiple human and mouse AML cell lines and primary AML cells. MCT4 is a plasma membrane H+and lactate co-transporter whose activity necessarily shifts protons extracellularly as intracellular lactate is extruded. MCT4 activity is increased when overexpressed or with increased intracellular lactate generated by glycolysis in the setting of nutrient abundance. With increased MCT4 activity, extracellular lactate and protons will increase causing extracellular acidification while alkalinizing the intracellular compartment. MCT4-knockout (MCT4-KO) of mouse and human AMLdid not induce compensatory MCT1 expression, reduced pHi, suppressed proliferation and improved animal survival. Growth reduction was experimentally defined to be due to intracellular acidification rather than lactate accumulation by independent modulation of those parameters. MCT4-KOmetabolic profiling demonstrated decreased ATP/ADP and increased NADP+/NADPH suggesting suppression of glycolysis and the pentose phosphate pathway (PPP) that was confirmed by stable isotopic carbon flux analyses. Notably,the enzymatic activity of purified gatekeeper enzymes, hexokinase 1 (HK1), pyruvate kinase M2 isoform (PKM2) and glucose-6-phosphate dehydrogenase (G6PDH) was sensitive to pH with increased activity at the leukemic pHi (pH 7.6) compared to normal pHi (pH 7.3). Evaluating MCT4 transcriptional regulation, we defined that activating histonemarks, H3K27ac and H3K4me3, were enriched at the MCT4 promoter region as were transcriptional regulators MLL1 and Brd4 by ChIP in AML compared with normal cells. Pharmacologic inhibition of Brd4 suppressed Brd4 and H3K27ac enrichment and MCT4 expression in AML and reduced leukemic cell growth. To determine whether MCT4 based pHi changes were sufficient to increase cell proliferation, we overexpressed MCT4 in normal HSPC and demonstrated in vivo increases in growth in conjunction with pHi alkalization. Some other cell types also were increased in their growth kinetics by MCT4 overexpression and pHi increase. Therefore, proton shifting may be a means by which cells respond to nutrient abundance, co-transporting lactate and protons out of the cell, increasing the activity of enzymes that enhance PPP and glycolysis for biomass generation. Epigenetic changes in AML appear to exploit that process by increasing MCT4 expression to enforce proton exclusion thereby gaining a growth advantage without dependence on signaling pathways. Inhibiting MCT4 and intracellular alkalization may diminish the ability of AML to outcompete normal hematopoiesis. Figure Disclosures Scadden: Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership.

scholarly journals EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1528-1528
Author(s):  
Sebastian Stasik ◽  
Jan Moritz Middeke ◽  
Michael Kramer ◽  
Christoph Rollig ◽  
Alwin Krämer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Employment Equity ◽  
Advisory Committees ◽  
Mutational Status ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Purpose: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and key epigenetic regulator involved in transcriptional repression and embryonic development. Loss of EZH2 activity by inactivating mutations is associated with poor prognosis in myeloid malignancies such as MDS. More recently, EZH2 inactivation was shown to induce chemoresistance in acute myeloid leukemia (AML) (Göllner et al., 2017). Data on the frequency and prognostic role of EZH2-mutations in AML are rare and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n = 1604) for EZH2 mutations. Patients and Methods: All patients analyzed had newly diagnosed AML, were registered in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML) and had available material at diagnosis. Screening for EZH2 mutations and associated alterations was done using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow or peripheral blood. Detection was conducted with a defined cut-off of 5% variant allele frequency (VAF). All samples below the predefined threshold were classified as EZH2 wild type (wt). Patient clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. Results: EZH2-mutations were found in 63 of 1604 (4%) patients, with a median VAF of 44% (range 6-97%; median coverage 3077x). Mutations were detected within several exons (2-6; 8-12; 14-20) with highest frequencies in exons 17 and 18 (29%). The majority of detected mutations (71% missense and 29% nonsense/frameshift) were single nucleotide variants (SNVs) (87%), followed by small indel mutations. Descriptive statistics of clinical parameters and associated co-mutations revealed significant differences between EZH2-mut and -wt patients. At diagnosis, patients with EZH2 mutations were significantly older (median age 59 yrs) than EZH2-wt patients (median 56 yrs; p=0.044). In addition, significantly fewer EZH2-mut patients (71%) were diagnosed with de novo AML compared to EZH2-wt patients (84%; p=0.036). Accordingly, EZH2-mut patients had a higher rate of secondary acute myeloid leukemia (sAML) (21%), evolving from prior MDS or after prior chemotherapy (tAML) (8%; p=0.036). Also, bone marrow (and blood) blast counts differed between the two groups (EZH2-mut patients had significantly lower BM and PB blast counts; p=0.013). In contrast, no differences were observed for WBC counts, karyotype, ECOG performance status and ELN-2017 risk category compared to EZH2-wt patients. Based on cytogenetics according to the 2017 ELN criteria, 35% of EZH2-mut patients were categorized with favorable risk, 28% had intermediate and 37% adverse risk. No association was seen with -7/7q-. In the group of EZH2-mut AML patients, significantly higher rates of co-mutations were detected in RUNX1 (25%), ASXL1 (22%) and NRAS (25%) compared to EZH2-wt patients (with 10%; 8% and 15%, respectively). Vice versa, concomitant mutations in NPM1 were (non-significantly) more common in EZH2-wt patients (33%) vs EZH2-mut patients (21%). For other frequently mutated genes in AML there was no major difference between EZH2-mut and -wt patients, e.g. FLT3ITD (13%), FLT3TKD (10%) and CEBPA (24%), as well as genes encoding epigenetic modifiers, namely, DNMT3A (21%), IDH1/2 (11/14%), and TET2 (21%). The correlation of EZH2 mutational status with clinical outcomes showed no effect of EZH2 mutations on the rate of complete remission (CR), relapse free survival (RFS) and overall survival (OS) (with a median OS of 18.4 and 17.1 months for EZH2-mut and -wt patients, respectively) in the univariate analyses. Likewise, the multivariate analysis with clinical variable such as age, cytogenetics and WBC using Cox proportional hazard regression, revealed that EZH2 mutations were not an independent risk factor for OS or RFS. Conclusion EZH mutations are recurrent alterations in patients with AML. The association with certain clinical factors and typical mutations such as RUNX1 and ASXL1 points to the fact that these mutations are associated with secondary AML. Our data do not indicate that EZH2 mutations represent an independent prognostic factor. Disclosures Middeke: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Scholl:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding. Brümmendorf:Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Merck: Consultancy; Pfizer: Consultancy, Research Funding. Burchert:AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding; Roche: Research Funding; Johnson & Johnson: Research Funding; Affimed: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

Detection of the JAK2V617F Mutation in Myeloproliferative Disorders by Melting Curve Analysis Using the LightCycler System

2006 ◽  
Vol 130 (7) ◽  
pp. 997-1003
Author(s):  
Randall J. Olsen ◽  
Zhouwen Tang ◽  
Daniel H. Farkas ◽  
David W. Bernard ◽  
Youli Zu ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Clinical Laboratory ◽  
Melting Curve ◽  
Myeloproliferative Disorder ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Wild Type ◽  
Acute Myeloid

Abstract Context.—A specific mutation, JAK2V617F, was recently recognized as having diagnostic value for myeloproliferative disorders. No practical assay is currently available for routine use in a clinical laboratory. Objective.—We report the development of a real-time polymerase chain reaction melting curve analysis assay that is appropriate for molecular diagnostics testing. Design.—Specific primers and fluorescence resonance energy transfer probes were designed, and patients with a previously diagnosed myeloproliferative disorder, de novo acute myeloid leukemia, or reactive condition were selected. The DNA was extracted from fresh and archived peripheral blood and bone marrow specimens, and real-time polymerase chain reaction melting curve analysis was performed on the LightCycler platform (Roche Applied Science, Indianapolis, Ind). Results.—The JAK2 region was successfully amplified, and wild-type amplicons were reproducibly discriminated from JAK2V617F amplicons. Titration studies using homozygous wild-type and mutant cell lines showed the relative areas under a melting curve were proportional to allele proportion, and the assay reliably detected one mutant in 20 total cells. JAK2V617F was identified in patients previously diagnosed with a myeloproliferative disorder or acute myeloid leukemia transformed from myeloproliferative disorder, whereas a wild-type genotype was identified in patients with reactive conditions or de novo acute myeloid leukemia. Conclusions.—These findings demonstrate the suitability of this assay for identifying JAK2V617F in a clinical laboratory setting. Furthermore, the semiquantitative detection of JAK2V617F in archived specimens provides a new tool for studying the prognostic significance of this mutation.

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

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
G. D. Bailey ◽  
L. Doolan ◽  
A. Baskar ◽  
L. C. Smith ◽  
C. H. Seedhouse
Keyword(s):  
Acute Myeloid Leukemia ◽  
Acute Myeloid Leukaemia ◽  
Myeloid Leukaemia ◽  
Myeloid Leukemia ◽  
Splice Variants ◽  
Wild Type ◽  
Significant Bias ◽  
Acute Myeloid

Abstract 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.

scholarly journals A point mutation in the granulocyte colony-stimulating factor receptor (G-CSF-R) gene in a case of acute myeloid leukemia results in the overexpression of a novel G-CSF-R isoform

Blood ◽  
1995 ◽  
Vol 85 (4) ◽  
pp. 902-911 ◽  
Author(s):  
F Dong ◽  
M van Paassen ◽  
C van Buitenen ◽  
LH Hoefsloot ◽  
B Lowenberg ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Point Mutation ◽  
Myeloid Leukemia ◽  
Receptor Gene ◽  
Splice Donor Site ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Cytoplasmic Region ◽  
Stimulating Factor ◽  
Acute Myeloid

A novel human granulocyte colony-stimulating factor (G-CSF) receptor isoform, designated SD, has been identified in which the distal C- terminal cytoplasmic region, previously shown to be essential for maturation signalling, is substituted by an altered C-terminus. The SD receptor has a high affinity for G-CSF and retains the membrane- proximal cytoplasmic region known to be sufficient for proliferative signalling. Nonetheless, the SD isoform lacks the ability to transduce growth signals in murine BAF3 cells and, in contrast to the wild-type G- CSF receptor, is scarcely capable of activating JAK2 kinase. Expression of SD receptor was found to be low in normal granulocytes, but was significantly increased in a patient with acute myeloid leukemia (AML). The leukemic cells of this patient harbour a point mutation in the SD splice donor site of the G-CSF receptor gene. These findings provide the first evidence that mutations in the G-CSF receptor gene can occur in certain cases of clinical de novo AML. The possible contribution of defective G-CSF receptor signalling to leukemogenesis is discussed.

Sign in / Sign up

Export Citation Format

Share Document