Two Novel Methods for Rapid Detection and Quantification of DNMT3A R882 Mutations in Acute Myeloid Leukemia
2014; Elsevier BV; Volume: 17; Issue: 2 Linguagem: Inglês
10.1016/j.jmoldx.2014.10.003
ISSN1943-7811
AutoresMelissa Mancini, Syed Khizer Hasan, Tiziana Ottone, Serena Lavorgna, Claudia Ciardi, Daniela F. Angelini, Francesca Agostini, Adriano Venditti, Francesco Lo‐Coco,
Tópico(s)Epigenetics and DNA Methylation
ResumoDNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at amino acid R882 in the methyltransferase domain of the gene. DNMT3A mutations have been reported to be stable during disease progression and are associated with unfavorable outcome in acute myeloid leukemia patients with normal karyotype. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarkers for minimal residual disease monitoring. We describe a new rapid diagnostic RT-PCR assay based on TauI restriction enzyme reaction to identify DNMT3A R882 mutations at diagnosis. In addition, we developed a sensitive and specific test based on peptide nucleic acid real-time PCR technology to monitor DNMT3A R882H mutation. We identified 24 DNMT3A R882H mutated patients out of 134 acute myeloid leukemia screened samples and we analyzed in these patients the kinetics of minimal residual disease after induction and consolidation therapy. This assay may be useful to better assess response to therapy in patients with acute myeloid leukemia bearing the DNMT3A R882H mutation. DNMT3A mutations represent one of the most frequent gene alterations detectable in acute myeloid leukemia with normal karyotype. Although various recurrent somatic mutations of DNMT3A have been described, the most common mutation is located at amino acid R882 in the methyltransferase domain of the gene. DNMT3A mutations have been reported to be stable during disease progression and are associated with unfavorable outcome in acute myeloid leukemia patients with normal karyotype. Because of their prognostic significance and high stability during disease evolution, DNMT3A mutations might represent highly informative biomarkers for minimal residual disease monitoring. We describe a new rapid diagnostic RT-PCR assay based on TauI restriction enzyme reaction to identify DNMT3A R882 mutations at diagnosis. In addition, we developed a sensitive and specific test based on peptide nucleic acid real-time PCR technology to monitor DNMT3A R882H mutation. We identified 24 DNMT3A R882H mutated patients out of 134 acute myeloid leukemia screened samples and we analyzed in these patients the kinetics of minimal residual disease after induction and consolidation therapy. This assay may be useful to better assess response to therapy in patients with acute myeloid leukemia bearing the DNMT3A R882H mutation. The DNMT3A gene is located on chromosome 2p23.3 and encodes for an enzyme with methyltransferase activity. DNMT3A acts as an epigenetic regulator of gene expression that catalyses DNA methylation at specific regulatory sequences by the addition of methyl groups to cytosine residues at CpG DNA islands. DNMT3A mainly consists of three structural domains: a zinc finger domain, a proline-tryptophan tryptophan-proline domain, and methyltransferase domain. The zinc finger domain is involved in protein-protein interaction with different partners, such as the transcription factors RP58 and Myc, the heterochromatin protein HP1, and the histone methyltransferase Suv39h1. The proline-tryptophan tryptophan-proline domain mediates the enzyme targeting to specific nucleic acid sequences, whereas the methyltransferase is responsible of enzymatic activity of the protein.1Chen T. Li E. Establishment and maintenance of DNA methylation patterns in mammals.Curr Top Microbiol Immunol. 2006; 301: 179-201PubMed Google Scholar, 2Hou H.A. Kuo Y.Y. Liu C.Y. Chou W.C. Lee M.C. Chen C.Y. Lin L.I. Tseng M.H. Huang C.F. Chiang Y.C. Lee F.Y. Liu M.C. Liu C.W. Tang J.L. Yao M. Huang S.Y. Ko B.S. Hsu S.C. Wu S.J. Tsay W. Chen Y.C. Tien H.F. DNMT3A mutations in acute myeloid leukemia: stability during disease evolution and clinical implications.Blood. 2012; 119: 559-568Crossref PubMed Scopus (183) Google Scholar, 3Roller A. Grossmann V. Bacher U. Poetzinger F. Weissmann S. Nadarajah N. Boeck L. Kern W. Haferlach C. Schnittger S. Haferlach T. Kohlmann A. Landmark analysis of DNMT3A mutations in hematological malignancies.Leukemia. 2013; 27: 1573-1578Crossref PubMed Scopus (60) Google Scholar Recently, Ley et al4Ley T.J. Ding L. Walter M.J. McLellan M.D. Lamprecht T. Larson D.E. et al.DNMT3A mutations in acute myeloid leukemia.N Engl J Med. 2010; 363: 2424-2433Crossref PubMed Scopus (1523) Google Scholar reported the occurrence of DNMT3A mutations (DNMT3Amut) in approximately 20% of patients with acute myeloid leukemia (AML). Of 281 AML patients, they identified >30 different somatic mutations scattered throughout the gene. Eighteen missense mutations, six frameshift, six nonsense, three splice-site mutations, and 1.5-Mb deletion that included DNMT3A and other genes were detected. Most DNMT3A mutations are located in the methyltransferase domain, and the most common is a missense mutation G>A at nucleotide position 2645, which leads to the substitution of an arginine with a histidine residue at amino acid position 882 (hereafter called DNMT3A R882H). Although the effect of mutations on enzyme activity is still poorly understood, it is has been hypothesized that recurrent mutations at a specific amino acid position may result in reduced enzymatic activity of the altered protein.4Ley T.J. Ding L. Walter M.J. McLellan M.D. Lamprecht T. Larson D.E. et al.DNMT3A mutations in acute myeloid leukemia.N Engl J Med. 2010; 363: 2424-2433Crossref PubMed Scopus (1523) Google Scholar, 5Yan X.J. Xu J. Gu Z.H. Pan C.M. Lu G. Shen Y. Shi J.Y. Zhu Y.M. Tang L. Zhang X.W. Liang W.X. Mi J.Q. Song H.D. Li K.Q. Chen Z. Chen S.J. Exome sequencing identified somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia.Nat Genet. 2011; 43: 309-315Crossref PubMed Scopus (621) Google Scholar, 6Shah M.Y. Licht J.D. DNMT3A mutations in acute myeloid leukemia.Nat Genet. 2011; 43: 289-290Crossref PubMed Scopus (51) Google Scholar DNMT3Amut occur at significantly higher frequency in AML patients with intermediate risk cytogenetic profile and M4/M5 morphologic findings, according to the French American British classification, are heterozygous, and are associated with other recurrent gene mutations, such as those affecting the FLT3-ITD, NPM1, and IDH1/2 genes. In addition, DNMT3Amut correlate in AML with some clinicobiological features, including older age and higher white blood cell and platelet counts.2Hou H.A. Kuo Y.Y. Liu C.Y. Chou W.C. Lee M.C. Chen C.Y. Lin L.I. Tseng M.H. Huang C.F. Chiang Y.C. Lee F.Y. Liu M.C. Liu C.W. Tang J.L. Yao M. Huang S.Y. Ko B.S. Hsu S.C. Wu S.J. Tsay W. Chen Y.C. Tien H.F. DNMT3A mutations in acute myeloid leukemia: stability during disease evolution and clinical implications.Blood. 2012; 119: 559-568Crossref PubMed Scopus (183) Google Scholar, 7Ribeiro A.F. Pratcorona M. Erpelinck-Verschueren C. Rockova V. Sanders M. Abbas S. Figueroa M.E. Zeilemaker A. Melnick A. Löwenberg B. Valk P.J. Delwel R. Mutant DNMT3A: a marker of poor prognosis in acute myeloid leukemia.Blood. 2012; 119: 5824-5831Crossref PubMed Scopus (200) Google Scholar, 8Gaidzik V.I. Schlenk R.F. Paschka P. Stölzle A. Späth D. Kuendgen A. von Lilienfeld-Toal M. Brugger W. Derigs H.G. Kremers S. Greil R. Raghavachar A. Ringhoffer M. Salih H.R. Wattad M. Kirchen H.G. Runde V. Heil G. Petzer A.L. Girschikofsky M. Heuser M. Kayser S. Goehring G. Teleanu M.V. Schlegelberger B. Ganser A. Krauter J. Bullinger L. Döhner H. Döhner K. Clinical impact of DNMT3A mutations in younger adult patients with acute myeloid leukemia: results of the AML study group (AMLSG).Blood. 2013; 121: 4769-4777Crossref PubMed Scopus (141) Google Scholar DNMT3Amut have been associated with poor clinical outcome in AML with an intermediate-risk cytogenetic profile even in multivariate analysis, and several studies have subsequently confirmed this prognostic role.2Hou H.A. Kuo Y.Y. Liu C.Y. Chou W.C. Lee M.C. Chen C.Y. Lin L.I. Tseng M.H. Huang C.F. Chiang Y.C. Lee F.Y. Liu M.C. Liu C.W. Tang J.L. Yao M. Huang S.Y. Ko B.S. Hsu S.C. Wu S.J. Tsay W. Chen Y.C. Tien H.F. DNMT3A mutations in acute myeloid leukemia: stability during disease evolution and clinical implications.Blood. 2012; 119: 559-568Crossref PubMed Scopus (183) Google Scholar, 9Im A.P. Sehgal A.R. Carroll M.P. Smith B.D. Tefferi A. Johnson D.E. Boyiadzis M. DNMT3A and IDH mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies.Leukemia. 2014; 28: 1774-1783Crossref PubMed Scopus (192) Google Scholar At the biological level, a recent study described the presence of DNMT3Amut in a preleukemic compartment of hematopoietic stem cells able to regenerate a complete hematopoietic hierarchy. Such a compartment led to clonal evolution and overt AML through its competitive advantage over the nonleukemic counterpart. Finally, this preleukemic compartment of DNMT3A mutated hematopoietic stem cells was also detected in remission samples, indicating its ability to survive intensive chemotherapy.10Shlush L.I. Zandi S. Mitchell A. Chen W.C. Brandwein J.M. Gupta V. Kennedy J.A. Schimmer A.D. Schuh A.C. Yee K.W. McLeod J.L. Doedens M. Medeiros J.J. Marke R. Kim H.J. Lee K. McPherson J.D. Hudson T.J. Brown A.M. Trinh Q.M. Stein L.D. Minden M.D. Wang J.C. Dick J.E. HALT Pan-Leukemia Gene Panel ConsortiumIdentification of pre-leukaemic haematopoietic stem cells in acute leukaemia.Nature. 2014; 506: 328-333Crossref PubMed Scopus (1033) Google Scholar Together, the above clinical and biological findings indicate that DNMT3Amut might represent relevant biomarkers to assess response to therapy in AML. We describe two novel assays for the detection and quantification of DNMT3Amut in AML patients. For the initial screening of the DNMT3Amut, we designed a simple and cost-effective diagnostic RT-PCR assay based on TauI restriction enzyme digestion followed by capillary electrophoresis. For the purpose of the quantitative analysis of the most common mutation (R882H) of DNMT3A, we developed a method based on peptide nucleic acid (PNA) real-time quantitative PCR (qPCR) in which the amplification of the wild-type (WT) allele of DNMT3A is inhibited and only the mutant allele is amplified. The PNAs are artificially synthesized DNA analogs in which the negatively charged sugar-phosphate backbone is replaced by a neutral peptidelike skeleton that consists of repeated N-(2-aminoethyl)-glycine units linked by peptide bonds. The PNA has favorable hybridization properties and biological stability because of its uncharged nature and peptide bond–linked backbone. The lack of electrostatic repulsion between PNA and DNA strands makes binding between PNA and DNA stronger than that between DNA and DNA.11Hyrup B. Nielsen P.E. Peptide nucleic acids (PNA): synthesis, properties and potential applications.Bioorg Med Chem. 1996; 4: 5-23Crossref PubMed Scopus (487) Google Scholar Recently, a PNA probe-based qPCR assay was developed for detection of the JAK2-V617F mutation.12Huijsmans C.J.J. Poodt J. Savelkoul P.H.M. Mirjam H.A. Hermans M.H.A. Sensitive detection and quantification of the JAK2V617F Allele by Real-Time PCR.J Mol Diagn. 2011; 13: 558-564Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar We evaluate the performance of PNA probe-based qPCR assay on the DNMT3A R882H monitoring in AML patients. Bone marrow samples were collected at diagnosis from 134 AML patients aged 18 to 60 years observed at the Institute of Hematology, Department of Biomedicine and Prevention, University Tor Vergata (Rome, Italy) between 2012 and 2014. Patients received intensive chemotherapy according to the European Organization of Research and Treatment of Cancer/Gruppo Italiano Malattie EMatologiche dell'Adulto protocols. For AML patients harboring DNMT3A R882H in their diagnostic samples, bone marrow specimens were also collected at different time points during treatment (after induction and consolidation therapy). Total RNA was extracted from Ficoll-Hypaque isolated bone mononuclear cells using standard procedures.13Chomczynski P. Sacchi N. Single step method of RNA isolation by acid guanidium thiocyanate phenol chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63148) Google Scholar RNA was reverse transcribed using random hexamers as primers.14van Dongen J.J. Macintyre E.A. Gabert J.A. Delabesse E. Rossi V. Saglio G. Gottardi E. Rambaldi A. Dotti G. Griensinger F. Parreira A. Gameiro P. Diaz M.G. Malec M. Langerak A.W. San Miguel J.F. Biondi A. Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease: report of the BIOMED-1 concerted action: investigation of minimal residual disease in acute myeloid leukemia.Leukemia. 1999; 13: 1901-1908Crossref PubMed Scopus (1006) Google Scholar Bone marrow RNAs were also obtained from 15 healthy marrow donors. All participants gave informed consent for the studies, and the investigation was approved by the Institutional Review Board at Policlinico Tor Vergata. For the diagnostic screening of DNMT3A R882 mutations, we used a strategy based on TauI enzyme RT-PCR assay followed by capillary electrophoresis. TauI recognizes and cuts the WT R882 sequence (GCSG∧C), whereas the presence of R882H, R882C, R882S, and R882P mutations abrogate the TauI cleavage site. A set of primers for exon 23 of the DNMT3A gene was designed. A forward primer (TauI_F1: 5′-TTTAAAGCCGCGGTATTTGGTTTCCCAGT-3′) harbored an additional TauI restriction site to control for the efficiency of the restriction enzyme reaction, whereas the reverse primer (TauI_R: 5′-6-FAM-CCTTACACACACGCAAAATACTCCTT-3′) was labeled with a fluorochrome to exploit capillary electrophoresis for the subsequent restriction fragment analysis. cDNA was amplified in a total volume of 25 μL of the reaction mixture containing 10 pmol of each primer, TauI_F1 and TauI_R, 1× PCR buffer, 2.5 mmol/L MgCl2, 5 mmol/L dNTPs, and 0.7U Taq polymerase (Applied Biosystems, Foster City, CA). Thermocycling conditions were as follows: 7 minutes at 95°C; 45 seconds at 95°C, 45 seconds at 66°C, and 45 seconds at 72°C for 35 cycles; and 7 minutes at 72°C. After amplification, 5 μL of the PCR product was digested with 0.6 μL of TauI (Thermo Fisher Scientific Inc, Waltham, MA) (3U/μL) and 1 μL of restriction buffer B (Thermo Fisher Scientific). Each digestion reaction was incubated at 55°C for 1 hour, and then the enzyme activity was blocked by the addition of 0.4 μL of 0.5 mol/L EDTA. One microliter of digested multiplex PCR product was mixed with deionized formamide and ROX500 size standard following the manufacturer's protocol, heated to 95°C for 5 minutes, and placed on ice for at least 1 minute before electrokinetic injection to the ABI 3130 capillary electrophoresis instrument (ABI 3130; Applied Biosystems). To confirm the results of the new enzymatic assay, we analyzed the exon 23 of DNMT3A gene by RT-PCR amplification followed by bidirectional direct DNA sequencing. We performed PCR using a DNMT3A_F: 5′-TTTATAAAGGACAGAAGATTCGGCAGAA-3′ and DNMT3A_R: 5′-CCTTACACACACGCAAAATACTCCTT-3′ as forward and reverse primers, respectively. Thermocycling conditions were as follows: 7 minutes at 95°C; 45 seconds at 95°C, 45 seconds at 58°C, 45 seconds at 72°C for 35 cycles; and 7 minutes at 72°C. PCR products were purified for sequencing using the Bigdye Terminator Cycle Sequencing Kit protocol version 3.1 (Applied Biosystems). Sequencing data were analyzed with SeqScape software version 2.5 (Applied Biosystems) to characterize the DNMT3A R882 mutations. To assess the detection threshold of our assay, we performed serial dilution experiments using RNAs obtained from the marrow of AML patients with >80% leukemic cells and harboring the DNMT3A R882H with RNAs extracted from the bone marrow of an AML patient with WT DNMT3A (mutant/WT ratio: 0.01, 0.10, 0.25, 0.50, 0.75, and 1.00). In a subset of 10 patients with DNMT3A R882H mutations, we compared the sensitivity of TauI restriction enzyme assay with the Sanger DNA sequencing using serially diluted samples. The qPCR assay for DNMT3A R882H quantification was designed using DNMT3A forward (DNMT3A_Fq: 5′-TGGTTTCCCAGTCCACTATACTGA-3′) and reverse primers (DNMT3A_Rq: 5′-TCCATGACCGGCCCAGCA-3′) and two different probes: a mutant fluorescent probe (DNMT3A R882H_probe: 5′-FAM-TCGCCAAGTGGCTCA-MGB-3′) with the identical sequence of the cDNA region containing the mutation and a WT probe (PNA_probe: 5′-Acetyl-CTCGCCAAGCGGCTCA-3′) altered with PNA to block WT allele amplification (Figure 1). The reverse primer and probes were designed using Primer Express software version 2.0 (Applied Biosystems). The qPCR assay was performed on an ABI Prism 7300 Sequence Detection System, containing a 96-well thermal cycler (SDS software version 1.2 analyses; Applied Biosystems). The reaction mixture (25 μL) contained 1× Master Mix (Applied Biosystems), 300 nmol/L of each primer, 200 nmol/L of DNMT3A R882H_probe, 1.6 μmol/L of PNA_probe (PanaGene, Daejeon, Korea; alternate source: Biosynthesis Inc., Lewisville, TX) and 5 μL of cDNA (1/10 of reverse transcription product). Amplification conditions were 2 minutes at 50°C, 10 minutes at 95°C, followed by 50 cycles at 95°C for 15 seconds and at 58°C for 1 minute. The threshold was set at 0.1, and baseline was set from cycles 3 to 15 for both reporters. We used ABL1 gene amplification as the internal control.15Gabert J. Beillard E. van der Velden V.H. Bi W. Grimwade D. Pallisgaard N. Barbany G. Cazzaniga G. Cayuela J.M. Cavé H. Pane F. Aerts J.L. De Micheli D. Thirion X. Pradel V. González M. Viehmann S. Malec M. Saglio G. van Dongen J.J. Standardization and quality control studies of 'real time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia- a Europe against cancer program.Leukemia. 2003; 17: 2318-2357Crossref PubMed Scopus (1228) Google Scholar We therefore evaluated the ability of DNMT3A R882H real-time PCR assay to discriminate the false-positive AML samples by analyzing 30 cDNA samples with DNMT3A WT. The DNMT3A R882H sensitivity and specificity tests were performed with serial water dilutions of mutated RNA and with RNA from healthy donors, respectively. Receiver operating characteristic (ROC) analysis was performed with MedCalc software version 14.12.0 (Osten, Belgium), and the SEM was calculated using the guidelines of DeLong et al.16DeLong E.R. DeLong D.M. Clarke-Pearson D.L. Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach.Biometrics. 1988; 44: 837-845Crossref PubMed Scopus (14161) Google Scholar The CI for the area under the curve (AUC) was calculated using a binomial exact CI. The RT-PCR amplified product of a DNMT3A R882H–positive case that was obtained using DNMT3A_F and DNMT3A_R was cloned into the plasmid vector pCR II-TOPO (Invitrogen, Groningen, the Netherlands). After colony selection, plasmid extracted with a Macherey Nagel Nucleospin Plasmid DNA purification kit from recombinant colonies was sequenced to verify whether a DNMT3A R882H mutation was present. Plasmid DNA concentration was determined by absorbance measurement, and five serial plasmid dilutions (106, 105, 103, 102, and 101 copies) were prepared for PNA qPCR assay to construct a standard curve for the quantitative assessment of DNMT3A R882H mutation copy number. The DNMT3A R882 mutations are located within a TauI restriction enzyme digestion site, and the mutations on R882 codon result in loss of this cut site (Figure 2A). Our RT-PCR assay, followed by TauI restriction enzyme digestion and separation and sizing by capillary electrophoresis, was performed in 134 AML diagnostic samples. A total of 29 cases (22%) had the DNMT3A R882 mutations (24 with R882H and 5 with R882C mutations), consistent with the results of previously published studies.4Ley T.J. Ding L. Walter M.J. McLellan M.D. Lamprecht T. Larson D.E. et al.DNMT3A mutations in acute myeloid leukemia.N Engl J Med. 2010; 363: 2424-2433Crossref PubMed Scopus (1523) Google Scholar, 8Gaidzik V.I. Schlenk R.F. Paschka P. Stölzle A. Späth D. Kuendgen A. von Lilienfeld-Toal M. Brugger W. Derigs H.G. Kremers S. Greil R. Raghavachar A. Ringhoffer M. Salih H.R. Wattad M. Kirchen H.G. Runde V. Heil G. Petzer A.L. Girschikofsky M. Heuser M. Kayser S. Goehring G. Teleanu M.V. Schlegelberger B. Ganser A. Krauter J. Bullinger L. Döhner H. Döhner K. Clinical impact of DNMT3A mutations in younger adult patients with acute myeloid leukemia: results of the AML study group (AMLSG).Blood. 2013; 121: 4769-4777Crossref PubMed Scopus (141) Google Scholar, 17F1 Thol Damm F. Lüdeking A. Winschel C. Wagner K. Morgan M. Yun H. Göhring G. Schlegelberger B. Hoelzer D. Lübbert M. Kanz L. Fiedler W. Kirchner G. Heil H. Krauter J. Ganser A. Heuser M. Incidence and prognostic influence of DNMT3A mutations in acute myeloid leukemia.J Clin Oncol. 2011; 29: 2889-2896Crossref PubMed Scopus (316) Google Scholar After capillary electrophoresis, all DNMT3A R882 WT patients revealed only one fragment of 93 bp as the result of enzymatic digestion, whereas DNMT3A R882 mutated patients invariably had two different fragments of 93 and 145 bp, in keeping with the heterozygous state of DNMT3A R882 mutations (Figure 2A). To assess the correct functioning of the digestion reaction, no residual fragment at 153 bp (nondigested fragment) should be detectable. A representative electropherogram of cases with and without DNMT3A R882 mutation is reported in Figure 2B. To validate the assay, all diagnostic samples were further analyzed by direct sequencing, which revealed concordance in 100% of cases. To establish the sensitivity of the TauI restriction enzyme digestion, serial dilution experiments were performed using RNAs from AML patients harboring the DNMT3A R882 mutation. We ran each dilution sample in duplicate on an ABI 3130 instrument (Applied Biosystems) (Supplemental Figure S1A), and the results indicated that this assay could detect the DNMT3A R882 mutations present in concentrations as low as 10%. To ensure the 10% limit of detection, a WT peak height of at least 2000 to 3000 arbitrary units was required together with a background noise not exceeding 20 arbitrary units. In 2 10 diluted samples, sequencing results revealed the presence of a mismatch on codon R882 of DNMT3A gene (Supplemental Figure S1B). In the remaining eight samples, the mismatch was unclear because of the background noise, whereas the TauI restriction enzyme assay clearly revealed the presence of DNMT3A mutations. The specificity and sensitivity of the PNA qPCR assay were evaluated by ROC curve analysis. To discriminate the false-positive AML samples, we inserted in the analysis the copy number of DNMT3A R882H obtained from an AML patient with the DNMT3A mutation and a pool of DNMT3A WT cDNA. Moreover, PNA qPCR results obtained from a serial dilution of mutated RNA samples with RNA from healthy donors were used for ROC analysis. This identified the value corresponding to the dilution of 10−4 as having a specificity and sensitivity of the test >93%. The AUC was calculated as 0.99, with a 95% CI ranging from 0.918 to 1.000 and a significance level of P < 0.0001 (Supplemental Figure S2). To analyze the kinetics of DNMT3A R882H, we quantified the copy number of the mutated allele at three different time points in 24 AML patients receiving chemotherapy. Every sample was set up in duplicate for each run. At diagnosis, the DNMT3A R882H median transcript level was 1101.5/104 ABL1 copies; after induction and consolidation therapy, the median transcript level decreased to 127/104 ABL1 copies and 76.5/104 ABL1 copies, respectively (Figure 3). A complete remission was achieved in 92% of patients after initial chemotherapy, whereas two had a positive minimal residual disease after induction and consolidation therapy. Of interest, these two samples maintained a high DNMT3A R882H copy number until after consolidation therapy. Quantitative analysis of DNMT3A R882H performed in 15 healthy donors revealed, as expected, no amplification in all cases. After serial dilution experiments, the assay revealed maximum reproducible sensitivity and specificity at 10−4. A representative standard curve plot of a PNA qPCR assay is shown in Supplemental Figure S3. We report two novel molecular assays for rapid diagnosis of DNMT3A R882mut and monitoring of the most common mutation affecting DNMT3A R882H. Both assays perform efficiently with satisfactory sensitivity and proved highly specific as indicated by the parallel sequencing analyses. Compared with DNA Sanger sequencing, which is commonly used to detect this aberration, our test appears less laborious and time-consuming as well as cost-effective and easily interpretable. Given the prognostic relevance of the DNMT3Amut in AML, the method may therefore conveniently replace the DNA Sanger sequencing in the routine molecular diagnostic workup of AML. A recent study based on the endonuclease restriction principle further supports the usefulness of enzymatic assay in detection of the DNMT3A R882H mutation.18Berenstein R. Blau I.W. Kar A. Cay R. Sindram A. Seide C. Blau O. Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia.J Exp Clin Cancer Res. 2014; 21: 33-44Google Scholar When comparing the high-resolution melting curve method with endonuclease restriction analysis, Berenstein et al18Berenstein R. Blau I.W. Kar A. Cay R. Sindram A. Seide C. Blau O. Comparative examination of various PCR-based methods for DNMT3A and IDH1/2 mutations identification in acute myeloid leukemia.J Exp Clin Cancer Res. 2014; 21: 33-44Google Scholar reported that high-resolution melting curve analysis had a lower sensitivity but, like our enzymatic assay, had the benefit of identifying different mutations in one PCR reaction. The simultaneous identification of all DNMT3A R882mut is relevant because all these mutations have an unfavorable prognostic effect on AML.2Hou H.A. Kuo Y.Y. Liu C.Y. Chou W.C. Lee M.C. Chen C.Y. Lin L.I. Tseng M.H. Huang C.F. Chiang Y.C. Lee F.Y. Liu M.C. Liu C.W. Tang J.L. Yao M. Huang S.Y. Ko B.S. Hsu S.C. Wu S.J. Tsay W. Chen Y.C. Tien H.F. DNMT3A mutations in acute myeloid leukemia: stability during disease evolution and clinical implications.Blood. 2012; 119: 559-568Crossref PubMed Scopus (183) Google Scholar, 9Im A.P. Sehgal A.R. Carroll M.P. Smith B.D. Tefferi A. Johnson D.E. Boyiadzis M. DNMT3A and IDH mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies.Leukemia. 2014; 28: 1774-1783Crossref PubMed Scopus (192) Google Scholar Regarding the PNA-based qPCR assay for DNMT3A R882H, our TauI restriction enzyme assay may prove useful to assess response to therapy at the molecular level in patients with AML bearing this mutation. The design of a quantitative assay for point mutations is a challenging task because of the need to develop primers and probes that have to accurately discriminate between WT and mutant alleles. Shivarov et al19Shivarov V. Ivanova M. Naumova E. Rapid detection of DNMT3A R882 mutations in Hematologic malignances using a novel bead-based suspension assay with BNA(NC) probes.PLoS One. 2014; 9: e99769Crossref PubMed Scopus (12) Google Scholar recently reported the applicability of bridged nucleic acid (BNA) probes coupled with fluorescently labeled beads for quantitative detection of DNMT3A R882 mutations. The difference between PNA- and BNA-based hybridization relies not only on probe labeling and the synthesis process but also on the platforms used for signal detection. The PNA-based strategy requires a real-time PCR instrument, whereas the BNA-based hybridization strategy needs the LabScan flow platform. Shivarov et al19Shivarov V. Ivanova M. Naumova E. Rapid detection of DNMT3A R882 mutations in Hematologic malignances using a novel bead-based suspension assay with BNA(NC) probes.PLoS One. 2014; 9: e99769Crossref PubMed Scopus (12) Google Scholar reported a 2.5% sensitivity of mutant allele detection using BNA in their DNMT3A R882 quantification assay. Another recent study found that DNMT3Amut appears as a founding mutation in preleukemic hematopoietic stem cells.10Shlush L.I. Zandi S. Mitchell A. Chen W.C. Brandwein J.M. Gupta V. Kennedy J.A. Schimmer A.D. Schuh A.C. Yee K.W. McLeod J.L. Doedens M. Medeiros J.J. Marke R. Kim H.J. Lee K. McPherson J.D. Hudson T.J. Brown A.M. Trinh Q.M. Stein L.D. Minden M.D. Wang J.C. Dick J.E. HALT Pan-Leukemia Gene Panel ConsortiumIdentification of pre-leukaemic haematopoietic stem cells in acute leukaemia.Nature. 2014; 506: 328-333Crossref PubMed Scopus (1033) Google Scholar The competitive growth advantage of the preleukemic pool of hemopoietic stem cells over the nonleukemic stem cells leads to clonal expansion and eventually AML evolution of the DNMT3A mutated stem cell compartment through the acquisition of additional genetic abnormalities. Therefore, the initial screening of AMLs for DNMT3Amut could be relevant for better stratification and management of AML patients. In addition, monitoring of DNMT3Amut through our PNA-based qPCR test during treatment may provide information on DNMT3Amut kinetics and may be informative in early prediction of disease evolution. In conclusion, considering the high cost and the relatively low sensitivity of the DNA sequencing method, the TauI restriction enzyme assay described herein represents a sensitive and specific method for DNMT3A R882mut routine screening especially in AML samples with low DNMT3A R882mut expression. In addition, considering that this method is simpler, more rapid, sensitive, and less expensive compared with DNA Sanger sequencing, it could be easily applicable in countries with limited resources. Further prospective longitudinal studies in large cohorts of AML patients are needed to investigate the clinical effect of DNMT3A R882mut on the outcome of the disease in terms of response to therapy and prediction of disease relapse. Download .jpg (.36 MB) Help with files Supplemental Figure S1Representative data of a DNMT3A R882 mutated sample diluted 10−1 with wild-type RNA. Electropherograms of RT-PCR product followed by a TauI restriction enzyme digestion of one sample run in duplicate (A) and sequencing chromatograms of diluted samples obtained using a reverse primer (B). Arrows indicate the mismatch on codon R882 of the DNMT3A gene GCG > GTG. Download .jpg (.58 MB) Help with files Supplemental Figure S2Receiver operating characteristic (ROC) curve for DNMT3A R882H real-time PCR assay with peptide nucleic acid blocking. As indicated by the asterisk, we used the method of Delong et al15Gabert J. Beillard E. van der Velden V.H. Bi W. Grimwade D. Pallisgaard N. Barbany G. Cazzaniga G. Cayuela J.M. Cavé H. Pane F. Aerts J.L. De Micheli D. Thirion X. Pradel V. González M. Viehmann S. Malec M. Saglio G. van Dongen J.J. Standardization and quality control studies of 'real time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia- a Europe against cancer program.Leukemia. 2003; 17: 2318-2357Crossref PubMed Scopus (1228) Google Scholar for the calculation of the SEM of the area under curve (AUC). As indicated by the dagger, CI for the AUC was calculated using the binomial exact CI. Download .jpg (.11 MB) Help with files Supplemental Figure S3Standard curve of DNMT3A R882H–positive plasmid dilutions (black data points with black trend line) plotted as cycle number (Ct) versus arbitrary transcript levels.
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