Quantitative detection of DNMT3A R882H mutation in acute myeloid leukemia

Background DNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia (AML) with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at R882 in the methyltransferase domain of the gene. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarkers for prognosis and outcome of disease. Methods We describe an allele-specific PCR with a Blocking reagent for the quantitative detection of DNMT3A R882H mutation providing the possibility to analyze the quantitative amount of mutation during the course of disease. Next, we analyzed 62 follow- up samples from 6 AML patients after therapy and allogeneic stem cell transplantation (alloSCT). Results We developed an ASB-PCR assay for quantitative analysis of R882H DNMT3A mutation. After optimization of blocker concentration, a R882H-positive plasmid was constructed to enhance the accuracy of the sensitivity of quantitative detection. The assay displayed a high efficiency and sensitivity up to 10−3. The reproducibility of assay analyzed using follow-up samples showed the standard deviation less than 3.1 %. This assay displayed a complete concordance with sequencing and endonuclease restriction analysis. We have found persistence of DNMT3A R882H mutations in complete remission (CR) after standard cytoreduction therapy that could be indicating presence of DNMT3A mutation in early pre-leukemic stem cells that resist chemotherapy. The loss of correlation between NPM1 and DNMT3A in CR could be associated with evolution of pre-leukemic and leukemic clones. In patients with CR with complete donor chimerism after alloSCT, we have found no DNMT3A R882H. In relapsed patients, all samples showed an increasing of both NPM1 and DNMT3A mutated alleles. This suggests at least in part the presence of NPM1 and DNMT3A mutations in the same cell clone. Conclusion We developed a rapid and reliable method for quantitative detection of DNMT3A R882H mutations in AML patients. Quantitative detection of DNMT3A R882H mutations at different time points of AML disease enables screening of follow-up samples. This could provide additional information about the role of DNMT3A mutations in development and progression of AML

Molecular Aberrations in Bone Marrow Stromal Cells in Multiple Myeloma

Multiple myeloma (MM) is a B-cell malignancy characterized by an accumulation of malignant plasma cells within the bone marrow. Bone marrow mesenchymal stromal cells (BMMSCs) represent a crucial component of MM microenvironment supporting its progression and proliferation. Alterations in BMMSC of MM (MM-BMMSC) have become an important research focus. In this study, we analyzed MM-BMMSC and their modification through interaction with plasma cells in 128 MM patients. MM-BMMSC displayed a senescence-like state that was accompanied by an increase in senescence-associated β-galactosidase activity, a reduced number of colony-forming units, an accumulation of cells in S phase of the cell cycle, and the overexpression of microRNAs (miR-16, miR-223, miR-485-5p, and miR-519d) and p21. MM-BMMSC showed a reduced expression of mitochondrial stress response protein SIRT3 and an increased mitochondrial DNA mass that led to a higher amount of reactive oxygen species compared to healthy donor BMMSC. The interaction between MM cells and MM-BMMSC is a complex mechanism that relies on multiple interacting signaling pathways. Observed aberrations in MM-BMMSC should be confirmed in an in vivo model in order to clarify the importance for the pathogenesis of MM. Eventually, the result of MM therapy could be improved by understanding the interaction between MM cells and MM-BMSCs

Tumor-stroma interactions in multiple myeloma

Multiple myeloma (MM) is a B-cell malignancy characterized by accumu- lation of malignant plasma cells (PC) within the bone marrow. Bone marrow mesenchymal stromal cells (BMMSCs) represent a crucial component of multiple myeloma (MM) microenvironment supporting its progression and proliferation. Alterations in bone marrow mesenchymal stromal cells of multiple myeloma patients (MM-BMMSCs) have become an important research focus. However, the role of alterations of MM-BMMSCs in the pathophysiology of MM is not clear. In this study, aberrations in MM-BMMSCs and their modification through interaction with MM cells should be analysed. MM-BMMSCs displayed a senescence-like state that was accompanied by an increased senescence associated ß-galactosidase activity (SAßGalA), a reduced number of colony-forming units, an accumulation of cells in S phase of the cell cycle and the overexpression of different microRNAs (miR-16, miR-223, miR-485-5p, miR-519d) and p21. MM-BMMSCs showed reduced expression of mitochondrial stress response protein SIRT3 and an increased mitochondrial DNA mass that was associated with a higher amount of reactive oxygen species compared to healthy donor BMMSCs (HD-BMMSCs). The overexpressed microRNAs miR-485-5p and miR-519d are located on DLK1-DIO3 and C19MC, respectively. Analyses revealed copy number accumulation and hypomethylation of both clusters. In vitro interaction with MM cells decreased the SAßGalA of MM-BMMSCs in correlation with the reduction of p21 and cells in S phase of the cell cycle. MiR-485-5p was significantly decreased in co-cultured MM-BMMSCs in connection with an increased methylation of DLK1-DIO3. Modification of miR-485-5p levels using microRNA mimic or inhibitor altered senescence and cell cycle characteristics of MM-BMMSCs. Cell contact to MM cells induced increased expression of SIRT3 associated with reduced levels of reactive oxygen species in MM-BMMSCs. Co-cultured MM-BMMSCs displayed an increased level of monocarboxylate transporter MCT1/CD147, whereas co-cultured MM cells showed a higher level of MCT4/CD147. In this context, interaction between MM-BMMSCs and MM cells led to the exchange of lactate, which was inhibited by treatment of co-cultures with MCT inhibitor alpha-cyano-4-hydroxycinnamic acid (alpha-CN). This induced apoptosis in MM cells and suppressed the increase of SIRT3 as well as the reinforced activation of NF-kappaB in MM-BMMSCs. Inhibition of SIRT3 in HD-BMMSCs using siRNA induced increased SAßGalA and a higher level of reactive oxygen species. Furthermore, knock-down of SIRT3 led to the accumulation of HD-BMMSCs in S phase of the cell cycle. MiR-223 expression in MM-BMMSCs was reduced by the presence of MM cells in vitro in a cell-contact dependent manner. Co-cultivation of MM cells and MM-BMMSCs induced activation of notch amongst others via jagged-2/notch-2 leading to increased expression of Hes1, Hey2 or Hes5 in both cell types. Cultivation of MM-BMMSCs with increasing levels of recombinant jagged-2 reduced miR-223 and increased Hes1 levels in a concentration-dependent manner. Transient reduction of miR-223 levels increased VEGF and IL-6 expression and secretion by MM-BMMSCs and impaired their osteogenic differentiation potential. Inhibition of notch signaling induced apoptosis in both MM cells and MM-BMMSCs. Furthermore, it increased miR-223 levels and reduced the expression of VEGF and IL-6 by both cell types. MM-BMMSCs show constitutive alterations in vitro resulting in a senescence-like state. MM cells modulate multiple pathways in senescence-like MM-BMMSCs leading to a better viability. SIRT3 and the DLK1-DIO3 cluster seem to influence the senescence-like state of MM-BMMSCs. Modulation of this phenotpye by MM cells can be reduced by treatment of co-cultures with MCT inhibitor alpha-CN. It is questionable whether the senescence-like state of MM-BMMSCs plays a pathological role in active MM or is more important for the promotion of a relapse. MiR-223 seems to participate in different MM supporting pathways in MM-BMMSCs including regulation of cytokine secretion as well as osteogenic differentiation. Further in vivo analysis regarding the role of the senescence-like state of MM-BMMSCs and of miR-223 for the support of MM could provide starting points for a more efficient anti-myeloma treatment.Das Multiple Myelom (MM) ist eine B-Zellneoplasie, welche sich durch die Akkumulation maligner Plasmazellen im Knochenmark klinisch manifestiert. Mesenchymale Knochenmarkstromazellen sind eine essentielle Komponente der Mikroumgebung des MM und können die Tumorproliferation und -progression fördern. Die Untersuchung von Alterationen in Knochenmarkstromazellen von MM-Patienten (MM-BMMSCs) stellt einen Schwerpunkt in der Erforschung des MM dar. Allerdings ist der Einfluss dieser Alterationen auf die Pathogenese des MM nicht vollständig geklärt. In dieser Arbeit sollten Aberrationen in MM-BMMSCs und deren Beeinflussung durch die Interaktion mit MM-Zellen untersucht werden. MM-BMMSCs zeigten einen seneszenzartigen Zustand, welcher sich durch die erhöhte Aktivität der Seneszenz-assoziierten ß-Galactosidase (SAßGal), die verringerte Anzahl von „colony-forming units“, die Akkumulation der Zellen in der S-Phase des Zellzyklus und die Überexpression verschiedener microRNAs (miR-16, miR-223, miR-485-5p, miR-519d) und p21 äußerte. Weiterhin war dieser Phänotyp von einer reduzierten Expression des mitochondrialen Stressproteins SIRT3, einer erhöhten Masse mitochondrialer DNA und einer erhöhten Menge an reaktiver Sauerstoffspezies im Vergleich zu BMMSCs von gesunden Spendern (HD-BMMSCs) begleitet. MiR-485-5p und miR-519d sind auf den zwei genomischen Clustern DLK1-DIO3 und C19MC lokalisiert. Analysen zeigten die Hypomethylierung und Kopieanzahlakkumulation von beiden Clustern in MM-BMMSCs. Die in vitro Interaktion mit MM-Zellen führte zur Erniedrigung der SAßGal-Aktivität, welche mit der reduzierten Expression von p21 und der Reduktion der Zellen in der S-Phase des Zellzyklus assoziiert war. MiR-485-5p war signifikant reduziert in co-kultivierten MM-BMMSCs in Verbindung mit einer gesteigerten Methylierung der regulatorischen Region von DLK1-DIO3. Die Modifizierung von miR-485-5p über microRNA Mimikry und Inhibitoren beeinflusste die Zellzykluseigenschaften und den seneszenzartigen Phänotyp von MM-BMMSCs. Des Weiteren verursachte der Zellkontakt zu MM-Zellen die erhöhte Expression von SIRT3 und die Reduktion der reaktiven Sauerstoffspezies in MM-BMMSCs. Zudem konnte in co-kultivierten MM-BMMSCs ein gesteigertes Level des Monocarboxylat-Transporters MCT1/CD147 detektiert werden, wogegen in co-kultivierten MM-Zellen MCT4/CD147 erhöht wurde. In diesem Zusammenhang zeigten MM-BMMSCs und MM-Zellen einen Austausch von Laktat, welcher durch die Behandlung von Co-Kulturen mit dem MCT-Inhibitor alpha-Cyano-4-hydroxyzimtsäure (alpha-CN) inhibiert wurde. Dies führte zur Apoptose der MM-Zellen, verhinderte die Steigerung von SIRT3 und die erhöhte Aktivierung des NF-kappaB Signalweges in MM-BMMSCs. Die Inhibierung der SIRT3 Expression in HD-BMMSCs mittels siRNA verursachte eine Steigerung der SAßGal-Aktivität, sowie des Gehalts an reaktiver Sauerstoffspezies, und induzierte die Akkumulation der HD-BMMSCs in der S-Phase des Zellzyklus. Zusätzlich zeigte sich bei Co-Kultivierung mit MM-Zellen eine zellkontakt- abhängige Reduktion von miR-223 in MM-BMMSCs. Die Co-Kultivierung verursachte die Aktivierung des Notch-Signalweges in beiden Zelltypen unter anderem über eine Jagged2-Notch2-Interaktion. Diese führte zur erhöhten Expression der Notch-Targetgene Hes1, Hey2 oder Hes5 in beiden Zellfraktionen. Die Inkubation von MM-BMMSCs mit rekombinantem Jagged2 verursachte die konzentrationsabhängige Steigerung von Hes1 und gleichzeitig die Reduktion von miR-223. Die transiente Inhibierung von miR-223 erhöhte die Sekretion und Expression von IL-6 und VEGF und verschlechterte das osteogene Differenzierungspotential von MM-BMMSCs. Die Inhibierung der Notch-Signalisierung verursachte Apoptose in MM-Zellen und MM-BMMSCs und reduzierte die Expression und Sekretion von VEGF und IL-6 beider Zelltypen. Gleichzeitig wurde der Gehalt von miR-223 in MM-BMMSCs gesteigert. MM-BMMSCs zeigen konstitutive Veränderungen in vitro, welche in einem seneszenzartigen Zustand resultieren. MM-Zellen modulieren verschiedene Signalwege in seneszenzartigen MM-BMMSCs, welche die Vitalität der Zellen zu verbessern scheinen. SIRT3 und der DLK1-DIO3 Cluster könnten einen Einfluss auf den seneszenzartigen Phänotyp von MM-BMMSCs haben. Die Beeinflussung dieses Phänotyps durch MM-Zellen kann durch den MCT-Inhibitor alpha-CN reduziert werden. Es ist fraglich ob dieser Phänotyp für die Pathogenese des aktiven MM von Bedeutung ist oder vielmehr ein Rezidiv des MM fördern könnte. MiR-223 scheint eine wichtige Rolle für multiple, tumorfördernde Signalwege in MM-BMMSCs zu spielen. Hierzu zählt die Modifikation der Zytokinsekretion und der osteogenen Differenzierung von MM-BMMSCs.Weiterführende in vivo Analysen bezüglich der Rolle des Seneszenz-artigen Phänotyps von MM-BMMSCs und von miR-223 für die Unterstützung des MM könnten neue Ansatzpunkte für eine effizientere Therapie des MM aufzeigen

Class III Receptor Tyrosine Kinases in Acute Leukemia – Biological Functions and Modern Laboratory Analysis

Acute myeloid leukemia (AML) is a complex disease caused by deregulation of multiple signaling pathways. Mutations in class III receptor tyrosine kinases (RTKs) have been implicated in alteration of cell signals concerning the growth and differentiation of leukemic cells. Point mutations, insertions, or deletions of RTKs as well as chromosomal translocations induce constitutive activation of the receptor, leading to uncontrolled proliferation of undifferentiated myeloid blasts. Aberrations can occur in all domains of RTKs causing either the ligand-independent activation or mimicking the activated conformation. The World Health Organization recommended including RTK mutations in the AML classification since their detection in routine laboratory diagnostics is a major factor for prognostic stratification of patients. Polymerase chain reaction (PCR)–based methods are well-validated for the detection of fms-related tyrosine kinase 3 ( FLT3 ) mutations and can easily be applied for other RTKs. However, when methodological limitations are reached, accessory techniques can be applied. For a higher resolution and more quantitative approach compared to agarose gel electrophoresis, PCR fragments can be separated by capillary electrophoresis. Furthermore, high-resolution melting and denaturing high-pressure liquid chromatography are reliable presequencing screening methods that reduce the sample amount for Sanger sequencing. Because traditional DNA sequencing is time-consuming, next-generation sequencing (NGS) is an innovative modern possibility to analyze a high amount of samples simultaneously in a short period of time. At present, standardized procedures for NGS are not established, but when this barrier is resolved, it will provide a new platform for rapid and reliable laboratory diagnostic of RTK mutations in patients with AML. In this article, the biological and physiological role of RTK mutations in AML as well as possible laboratory methods for their detection will be reviewed

Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia

BACKGROUND: Mutations in epigenetic modifiers were reported in patients with acute myeloid leukaemia (AML) including mutations in DNA methyltransferase 3A gene (DNMT3A) in 20%-30% patients and mutations in isocitrate dehydrogenase 1/2 gene (IDH1/2) in 5%-15% patients. Novel studies have shown that mutations in DNMT3A and IDH1/2 influence prognosis, indicating an increasing need to detect these mutations during routine laboratory analysis. DNA sequencing for the identification of these mutations is time-consuming and cost-intensive. This study aimed to establish rapid screening tests to identify mutations in DNMT3A and IDH1/2 that could be applied in routine laboratory procedures and that could influence initial patient management. METHODS: In this study we developed an endonuclease restriction method to identify the most common DNMT3A mutation (R882H) and an amplification-refractory mutation system (ARMS) to analyse IDH2 R140Q mutations. Furthermore, we compared these methods with HRM analysis and evaluated the latter for the detection of IDH1 mutations. RESULTS: Of 230 samples from patients with AML 30 (13%) samples had DNMT3A mutations, 16 (7%) samples had IDH2 R140Q mutations and 36 (16%) samples had IDH1 mutations. Sensitivity assays performed using serial dilutions of mutated DNA showed that ARMS analysis had a sensitivity of 4.5%, endonuclease restriction had a sensitivity of 0.05% and HRM analysis had a sensitivity of 5.9%–7.8% for detecting different mutations. HRM analysis was the best screening method to determine the heterogeneity of IDH1 mutations. Furthermore, for the identification of mutations in IDH2 and DNMT3A, endonuclease restriction and ARMS methods showed a perfect concordance (100%) with Sanger sequencing while HRM analysis showed a near-perfect concordance (approximately 98%). CONCLUSION: Our study suggested that all the developed methods were rapid, specific and easy to use and interpret. HRM analysis is the most timesaving and cost-efficient method to rapidly screen all the 3 genes at diagnosis in samples obtained from patients with AML. Endonuclease restriction and ARMS assays can be used separately or in combination with HRM analysis to obtain more reliable results. We propose that early screening of mutations in patients with AML having normal karyotype could facilitate risk stratification and improve treatment options