Rapid Detection of KIT Mutations in Core-Binding Factor Acute Myeloid Leukemia Using High-Resolution Melting Analysis
2009; Elsevier BV; Volume: 11; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2009.090043
ISSN1943-7811
AutoresÓscar Fuster, Eva Barragán, Pascual Bolufer, José Cervera, María‐José Larráyoz, Antonio Jiménez‐Velasco, Joaquín Martínez‐López, Ana Valencia, Federico Moscardó, Miguel Á. Sanz,
Tópico(s)Chronic Myeloid Leukemia Treatments
ResumoThe most frequent KIT mutations reported in core-binding factor acute myeloid leukemia are point mutations and insertions/deletions in exons 17 and 8. The vast majority of KIT mutation detection procedures are time-consuming, costly, or with a high lower limit of detection. High-resolution melting (HRM) is a gene scanning method that combines simplicity and rapid identification of genetic variants. We describe an HRM method for the simultaneous screening of exons 8 and 17 KIT mutations and report the results obtained in 69 core-binding factor acute myeloid leukemia patients. Mutation detection was compared with sequencing as the gold standard. The HRM method used high-resolution melting master reagents (Roche) and the LightCycler 480 (Roche) platform. HRM was reproducible, showed a lower limit of detection of 1%, and discriminated all patients with mutated KIT from controls without false positive or false negative results. Additionally, most of the mutations were differentiated from the other mutations. KIT mutations were present in 15.9% of patients, showing a higher incidence in inv(16) (25.8%) than in t(8;21) (7.9%). The presence of a KIT mutation was associated with a high white blood cell count, and adult patients with an exon 17 mutation had a higher incidence of relapse. These findings verify that HRM is a reliable, rapid, and sensitive method for KIT mutation screening. Furthermore, our study corroborates the unfavorable prognosis associated with exon 17 KIT mutations. The most frequent KIT mutations reported in core-binding factor acute myeloid leukemia are point mutations and insertions/deletions in exons 17 and 8. The vast majority of KIT mutation detection procedures are time-consuming, costly, or with a high lower limit of detection. High-resolution melting (HRM) is a gene scanning method that combines simplicity and rapid identification of genetic variants. We describe an HRM method for the simultaneous screening of exons 8 and 17 KIT mutations and report the results obtained in 69 core-binding factor acute myeloid leukemia patients. Mutation detection was compared with sequencing as the gold standard. The HRM method used high-resolution melting master reagents (Roche) and the LightCycler 480 (Roche) platform. HRM was reproducible, showed a lower limit of detection of 1%, and discriminated all patients with mutated KIT from controls without false positive or false negative results. Additionally, most of the mutations were differentiated from the other mutations. KIT mutations were present in 15.9% of patients, showing a higher incidence in inv(16) (25.8%) than in t(8;21) (7.9%). The presence of a KIT mutation was associated with a high white blood cell count, and adult patients with an exon 17 mutation had a higher incidence of relapse. These findings verify that HRM is a reliable, rapid, and sensitive method for KIT mutation screening. Furthermore, our study corroborates the unfavorable prognosis associated with exon 17 KIT mutations. Activating mutations in tyrosine kinases, FLT3, KRAS, NRAS, and KIT, are found in more than 30% of patients with de novo acute myeloid leukemia (AML).1Omasson MH Xiang Z Walgren R Zhao Y Kasai Y Miner T Ries RE Lubman O Fremont DH McLellan MD Payton JE Westervelt P DiPersio JF Link DC Walter MJ Graubert TA Watson M Baty J Heath S Shannon WD Nagarajan R Bloomfield CD Mardis ER Wilson RK Ley TJ Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukaemia.Blood. 2008; 111: 4797-4808Crossref PubMed Scopus (179) Google Scholar These tyrosine kinase mutations confer a proliferative or survival advantage to blastic cells and may cooperate with mutated transcription factors such as CBF complex, RARa, and MLL, by interfering with differentiation and contributing to the AML phenotype.2Renneville A Roumier C Biggio V Nibourel O Boissel N Fenaux P Preudhomme C Cooperating gene mutations in acute myeloid leukemia: a review of the literature.Leukemia. 2008; 22: 915-931Crossref PubMed Scopus (294) Google Scholar KIT gene encodes a transmembrane glycoprotein receptor that is member of the type III receptor tyrosine kinase family. The structure of this receptor consists of three domains: an extracellular domain composed of five immunoglobulin-like motives, a transmembrane and juxtamembrane domain, and an intracellular tyrosine kinase domain.3Paschka P Marcucci G Ruppert AS Mrózek K Chen H Kittles RA Vukosavljevic T Perrotti D Vardiman JW Carroll AJ Kolitz JE Larson RA Bloomfield CD Cancer and Leukemia Group B Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;16).J Clin Oncol. 2006; 24: 3904-3911Crossref PubMed Scopus (545) Google Scholar,4Lennartsson J Jelacic T Linnekin D Shivakrupa R Normal and oncogenic forms of the receptor tyrosine kinase kit.Stem Cells. 2005; 23: 16-43Crossref PubMed Scopus (236) Google Scholar After stem cell factor binding to the KIT receptor, the receptor dimerizes and becomes phosphorylated, activating the downstream signaling pathways implicated in cell proliferation, differentiation, and survival.4Lennartsson J Jelacic T Linnekin D Shivakrupa R Normal and oncogenic forms of the receptor tyrosine kinase kit.Stem Cells. 2005; 23: 16-43Crossref PubMed Scopus (236) Google Scholar Ligand-independent activation of KIT receptor may be achieved by gain-of-function mutations reported in AML as well as in several other malignancies. Activating KIT mutations are most frequently located within exon 17, which encodes the KIT activation loop in the kinase domain, and exon 8, which encodes a region of the extracellular domain of the KIT receptor.3Paschka P Marcucci G Ruppert AS Mrózek K Chen H Kittles RA Vukosavljevic T Perrotti D Vardiman JW Carroll AJ Kolitz JE Larson RA Bloomfield CD Cancer and Leukemia Group B Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;16).J Clin Oncol. 2006; 24: 3904-3911Crossref PubMed Scopus (545) Google Scholar,5Cairoli R Beghini A Grillo G Nadali G Elice F Ripamonti CB Colapietro P Nichelatti M Pezzetti L Lunghi M Cuneo A Viola A Ferrara F Lazzarino M Rodeghiero F Pizzolo G Larizza L Morra E Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study.Blood. 2006; 107: 3463-3468Crossref PubMed Scopus (302) Google Scholar,6Schnittger S Kohl TM Haferlach T Kern W Hiddemann W Spiekermann K Schoch C KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival.Blood. 2006; 107: 1791-1799Crossref PubMed Scopus (316) Google Scholar The most frequent KIT mutations reported are point mutations at codon 816 of exon 17 and insertions/deletions in exon 8. KIT mutations have been reported to occur preferentially in AML with core-binding factor (CBF) rearrangements such as RUNX1-RUNX1T1 and CBFb-MYH11, caused by the t(8;21) and inv(16) or t(16;16), hereafter abbreviated as inv(16). According to different reports, the incidence of KIT mutations in these malignancies varies widely from 6% to 46%.5Cairoli R Beghini A Grillo G Nadali G Elice F Ripamonti CB Colapietro P Nichelatti M Pezzetti L Lunghi M Cuneo A Viola A Ferrara F Lazzarino M Rodeghiero F Pizzolo G Larizza L Morra E Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study.Blood. 2006; 107: 3463-3468Crossref PubMed Scopus (302) Google Scholar6Schnittger S Kohl TM Haferlach T Kern W Hiddemann W Spiekermann K Schoch C KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival.Blood. 2006; 107: 1791-1799Crossref PubMed Scopus (316) Google Scholar7Shimada A Taki T Tabuchi K Tawa A Horibe K Tsuchida M Hanada R Tsukimoto I Hayashi Y KIT mutations, and not FLT3 internal tandem duplication, are strongly associated with a poor prognosis in pediatric acute myeloid leukemia with t(8;21): a study of the Japanese Childhood AML Cooperative Study Group.Blood. 2006; 107: 1806-1809Crossref PubMed Scopus (169) Google Scholar8Care RS Valk PJ Goodeve AC Abu-Duhier FM Geertsma-Kleinekoort WM Wilson GA Gari MA Peake IR Löwenberg B Reilly JT Incidence and prognosis of c-KIT and FLT3 mutations in core binding factor (CBF) acute myeloid leukaemias.Br J Haematol. 2003; 121: 775-777Crossref PubMed Scopus (274) Google Scholar Several studies have shown an adverse prognostic value for exon 17 KIT mutation in CBF AML3Paschka P Marcucci G Ruppert AS Mrózek K Chen H Kittles RA Vukosavljevic T Perrotti D Vardiman JW Carroll AJ Kolitz JE Larson RA Bloomfield CD Cancer and Leukemia Group B Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;16).J Clin Oncol. 2006; 24: 3904-3911Crossref PubMed Scopus (545) Google Scholar,5Cairoli R Beghini A Grillo G Nadali G Elice F Ripamonti CB Colapietro P Nichelatti M Pezzetti L Lunghi M Cuneo A Viola A Ferrara F Lazzarino M Rodeghiero F Pizzolo G Larizza L Morra E Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study.Blood. 2006; 107: 3463-3468Crossref PubMed Scopus (302) Google Scholar,6Schnittger S Kohl TM Haferlach T Kern W Hiddemann W Spiekermann K Schoch C KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival.Blood. 2006; 107: 1791-1799Crossref PubMed Scopus (316) Google Scholar; however, the relevance of exon 8 mutations still remains unknown. Various sensitive methods have been used to detect KIT mutations in CBF AML, such as polymerase chain reaction (PCR) followed by the screening of heteroduplex by high-performance liquid chromatography,3Paschka P Marcucci G Ruppert AS Mrózek K Chen H Kittles RA Vukosavljevic T Perrotti D Vardiman JW Carroll AJ Kolitz JE Larson RA Bloomfield CD Cancer and Leukemia Group B Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;16).J Clin Oncol. 2006; 24: 3904-3911Crossref PubMed Scopus (545) Google Scholar,5Cairoli R Beghini A Grillo G Nadali G Elice F Ripamonti CB Colapietro P Nichelatti M Pezzetti L Lunghi M Cuneo A Viola A Ferrara F Lazzarino M Rodeghiero F Pizzolo G Larizza L Morra E Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study.Blood. 2006; 107: 3463-3468Crossref PubMed Scopus (302) Google Scholar,9Goemans BF Zwaan CM Miller M Zimmermann M Harlow A Meshinchi S Loonen AH Hählen K Reinhardt D Creutzig U Kaspers GJ Heinrich MC Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia.Leukemia. 2005; 19: 1536-1542Crossref PubMed Scopus (209) Google Scholar sequencing,10Wang YY Zhou GB Yin T Chen B Shi JY Liang WX Jin XL You JH Yang G Shen ZX Chen J Xiong SM Chen GQ Xu F Liu YW Chen Z Chen SJ AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec.Proc Natl Acad Sci USA. 2005; 102: 1104-1109Crossref PubMed Scopus (268) Google Scholar and real-time PCR using hybridization probes.6Schnittger S Kohl TM Haferlach T Kern W Hiddemann W Spiekermann K Schoch C KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival.Blood. 2006; 107: 1791-1799Crossref PubMed Scopus (316) Google Scholar However, these methods are expensive, time-consuming, or have a reduced sensitivity, not allowing the detection of these mutations in samples with low blast cell count. The recent development of platforms and software for high-resolution melting (HRM) analysis11Wittwer CT Reed GH Gundry CN Vandersteen JG Pryor RJ High-resolution genotyping by amplicon melting analysis using LCGreen.Clin Chem. 2003; 49: 853-860Crossref PubMed Scopus (1020) Google Scholar has provided the introduction of this technology in the clinical laboratory. The great advantages of HRM (sensitivity, rapidity, simplicity, low cost, and high throughput) have justified the widespread use of these methods for detection of many small genetic variations.12Vaughn CP Elenitoba-Johnson KSJ High-Resolution melting analysis for detection of internal tandem duplications.J Mol Diagn. 2004; 6: 211-216Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar13Jones AV Cross NC White HE Green AR Scot LM Rapid identification of JAK2 exon 12 mutations using high resolution melting analysis.Haematologica. 2008; 93: 1560-1564Crossref PubMed Scopus (44) Google Scholar14Rapado I Grande S Albizua E Ayala R Hernández JA Gallardo M Gilsanz F Martinez-Lopez J High resolution melting analysis for JAK2 exon 14 and exon 12 mutations: a diagnostic tool for myeloproliferative neoplasms.J Mol Diagn. 2009; 11: 155-161Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar We report the results obtained in a group of 69 CBF AML patients by using a fast, sensitive, and reliable HRM method that allows the simultaneous screening of exons 8 and 17 KIT mutations. The study includes 69 CBF AML patients recruited between 1998 and 2008: 55 adults [30 with t(8;21), 24 with inv(16), and one with t(16;16)] and 14 children [8 with t(8;21) and six with inv(16)], who were diagnosed in four Spanish institutions. The leukemia subtypes were established according to the morphological and cytochemical criteria of the French-American-British (FAB) classification.15Benett JM Catovsky D Daniel MT Flandrin G Galton DAG Gralnick HR Sultan C Proposed revised criteria for the classification of acute myeloid leukemia: a report of the French-American-British Cooperative Group.Br J Haematol. 1976; 33: 451-458Crossref PubMed Scopus (4689) Google Scholar The presence of t(8;21) or inv(16) was detected by standard cytogenetics and the corresponding rearrangements, RUNX1-RUNX1T1 and CBFb-MYH11, by reverse transcription quantitative PCR (RQ-PCR). Patients received intensive chemotherapy following the PETHEMA LMA 96, LMA 99, and LMA 2006 protocols for adults in which induction therapy consisted of standard combinations of anthracycline plus cytarabine with or without etoposide. Children were treated according the SHOP 2001 protocol, which consisted of induction therapy with standard combinations of idarubicine plus cytarabine with or without etoposide. The Institutional Ethics Committee for Clinical Research approved this study, and informed consent was obtained from all patients and conducted in accordance with the recommendations of the Declaration of Human Rights, the Conference of Helsinki, and institutional regulations. KIT mutation study of exons 8 and 17 was performed on genomic DNA extracted from bone marrow samples collected during active disease (diagnosis and relapse). Genomic DNA was directly extracted from 500 μl of bone marrow using MagNA Pure LC DNA isolation kit large volume (Roche, Mannheim, Germany) automated in the MagNA Pure LC system (Roche). The quality and concentration of DNA extracted was assessed with a NanoDrop-1000 (NanoDrop Technologies Inc., Wilmington, DE). All samples were tested in duplicate using LightCycler 480 high resolution melting master (Roche Diagnostics, Mannheim, Germany) x1 containing Taq, nucleotides, and the dye ResoLight. Two DNA normal controls and one positive mutated DNA sample for each exon were included in each run. The HRM assays were performed using the LightCycler 480 instrument (Roche Diagnostics, Roche Instrument Center AG, Rotkreuz, Switzerland). Thirty nanograms of DNA were amplified in a 10-μl final volume containing 1X LightCycler 480 high resolution melting master (Roche), 0.5 μmol/L each primer pair (KIT F 5′-GCTGAGGTTTTCCAGCACTC-3′ and KIT R 5′-AATTGCAGTCCTTCCCCTCT-3′), and 2.0 mmol/L MgCl2 for exon 8 or 0.2 μmol/L of each primer pair (KIT S 5′-CAGCCAGAAATATCCTCCTTACT-3′ and KIT B 5′-TTGCAGGACTGTCAAGCAGAG-3′) and 3 mmol/L MgCl2 for exon 17. The expected size of the PCR product was 219 and 137 bp for exons 8 and 17, respectively. The PCR program consists of an initial preheating at 95°C for 10 minutes to activate the Taq DNA polymerase, followed by 45 amplification cycles. Each cycle comprises an annealing step at 56°C for 13 seconds, an elongation step at 72°C for 14 seconds, and denaturation at 94°C for 10 seconds. The final melting program consists of three main steps beginning with a denaturation at 95°C for 1 minute, renaturation at 40°C for 1 minute, and melting from 60°C to 95°C, with a ramp of 0.02°C per second and 25 fluorescence acquisitions per degree centigrade. Results were analyzed as fluorescence versus temperature graphs using Gene Scanning software version 1.5.0 (Roche Instrument Center, Rotkreuz, Switzerland). The melting curve analysis comprises three steps: normalization of melting curves, equaling to 100% the initial fluorescence and to 0% the fluorescence remnant after DNA dissociation; shifting of the temperature axis of the normalized melting curves to the point where the entire double-stranded DNA is completely denatured; and finally, the difference plot analyzing the differences in melting curve shape clustering the samples into groups based on the internal software calculation.12Vaughn CP Elenitoba-Johnson KSJ High-Resolution melting analysis for detection of internal tandem duplications.J Mol Diagn. 2004; 6: 211-216Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar The samples were directly sequenced from the LightCycler PCR amplification. The PCR products were mixed with ExoSap-IT (USB Corporation, Cleveland, OH) to remove the remaining primers and bidirectionally sequenced with forward and reverse primers using ABI PRISM terminator cycle sequencing kit version 1.1 (Applied Biosystems, Foster City, CA) on the ABI PRISM 3130 genetic analyzer (Applied Biosystems, Foster City, CA). The association of the presence of KIT mutation with the biological and demographic characteristics of leukemias was proved using the χ2 and Fisher's exact test. Unadjusted time-to-event analyses were performed using the Kaplan-Meier estimate16Kaplan EL Meier P Nonparametric estimations from incomplete observations.J Am Stat Assoc. 1958; 53: 457-481Crossref Scopus (48341) Google Scholar and for comparisons the log-rank test.17Mantel N Evaluation of survival data and two new rank order statistics arising in its consideration.Cancer Chemother Rep. 1966; 50: 163-170PubMed Google Scholar Disease-free survival (DFS) and relapse-free survival (RFS) were estimated from the date of complete remission. In the analysis of DFS, relapse and death in complete remission were considered uncensored events, whichever occurred first. For RFS, relapse in complete remission was considered the uncensored event. Patient follow-up was updated on June 30, 2008. All tests were two-sided, and a P value less than 0.05 was considered statistically significant. Computations were performed using the SPSS version 12.0 statistical package (Chicago, IL). Sixty-nine patients were screened for the presence of KIT mutations (67 at diagnosis and two relapses). The minigel electrophoresis of PCR products showed a single fragment with the expected size of 219 bp for exon 8 and 137 bp for exon 17. The melting analysis revealed the presence of a single peak with a mean melting temperature of 83.7°C and 79.5°C for exons 8 and 17, respectively. After normalization and temperature shift a clear difference between the group of wild-type DNA and the rest of the samples was observed. This difference was further enhanced with the difference plot, which establishes clusters of samples (Figure 1A and 1B). Hence, four positive samples in exon 8 and eight positive samples in exon 17 showed a clearly distinctive shape of the difference curve plot compared with those of the wild-type DNA clustered around baseline, thus allowing the distinction of these mutations. It should be noted that fluorescence curves obtained for the duplicates of each sample were practically superimposable either in the same assay or between assays. Patients' results were confirmed by sequencing directly from PCR products. Direct sequencing evidenced that all of the samples of exons 8 and 17 clustered around baseline as wild-type had normal DNA sequence, and the 11 patients classified in different clusters showed sequence variations/mutations. Considering the 69 samples analyzed by HRM, no false positive or negative results were found, yielding both sensitivity and specificity equal to 100%. We identified three patients with insertion/deletions in exon 8, seven patients with point mutations in exon 17, and one patient who presented mutations in both exons. In exon 8 we found the mutations T417_D419delinsRG, T417_V422delinsSRIL, T417_D419delinsS and T417_ D419delinsG. Moreover, in exon 17 we found two D816V, two D816Y, two D816H, one D820G, and one N822K mutation (Table 1).Table 1Cytogenetic and KIT Mutations Found in Exons 8 and 17ExonNo. of patientsCytogeneticsNucleotide changeMutation description81inv (16)del cttacgac ins gggggT417_D419delinsRG1inv (16)del acttacgacaggctcg ins tcgaggatctT417_V422delinsSRIL1inv (16)del ttacga ins gtcT417_D419delinsS1inv (16)del ttacga ins cggT417_D419delinsG172t (8;21), inv (16)gac → cacD816H2inv (16)gac → gtcD816V2inv (16)gac → tacD816Y1t (8;21)gat → ggtD820G1t (8;21)aat → aagN822K Open table in a new tab To determine the minimum detectable percentage of KIT mutated-cell DNA we serially diluted positive samples T417_D419delinsS for exon 8 and D820G for exon 17, which presented blast cell count close to 100% (1:10, 1:100, 1:300, 1:500, and 1:1000) in wild-type DNA. After performing the PCR assay we could still detect mutations for both exons up to dilution 1:100 (Figure 2A and 2B). The presence of KIT mutations was detected in 11 of 69 AML patients (15.9%; one infant and 10 adults); according to cytogenetic findings, three of 38 patients (7.9%) with t(8;21) and eight of 31 patients (25.8%) with inv(16) (P = 0.043). KIT mutations were associated with a high white blood cell count (P = 0.012). Thus, KIT mutated patients showed median values of white blood cells of 103*103/μl versus 20*103/μl of patients negative for KIT mutations. No association was found with age and gender. Complete remission was achieved in 87% of patients. No association was found between KIT mutations and response to induction chemotherapy. The median follow-up of adult patients was 17 months (range, 1–88 months) with estimated probabilities of DFS and RFS of 55% and 58%, respectively. The presence of KIT mutations in exons 8 or 17 did not influence DFS or RFS in adult patients. However, isolated exon 17 KIT mutations significantly influenced RFS (P = 0.04), although they did not modify DFS. Thus, the estimated 17-month RFS was 59% for non-mutated patients (n = 31), whereas it was 27% for patients with exon 17 mutated (n = 7) (Figure 3). With regard to the cytogenetic groups t(8;21) or inv (16), no differences were found in RFS or DFS. Our data support the finding that the HRM method here established allows the distinction of wild-type DNA from other DNA harboring KIT mutations in exons 8 and 17. Thus the differential plot clearly distinguishes the group of wild-type DNA samples from that with KIT mutations of AML patients. In this regard, the method is specific and sensitive enough for KIT mutation screening, since no false positive or negative results were found. Moreover, most of the mutations were well differentiated between them according to differential plot shapes, with the only exception of D816Y and D820G mutations that both were classified in the same group. In any case, all samples screened as positive for KIT mutations could be confirmed and characterized, in most cases by direct sequencing of PCR products of the HRM.13Jones AV Cross NC White HE Green AR Scot LM Rapid identification of JAK2 exon 12 mutations using high resolution melting analysis.Haematologica. 2008; 93: 1560-1564Crossref PubMed Scopus (44) Google Scholar However, in some cases we used locked nucleic acid oligonucleotides to sequence PCR products (data not shown)18Sotlar K Escribano L Landt O Möhrle S Herrero S Torrelo A Lass U Horny HP Bültmann B One-Step detection of c-KIT Point mutations using peptide nucleic acid-mediated polymerase chain reaction clamping and hybridization probes.Am J Pathol. 2003; 162: 737-746Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar19Wallén M Tomás E Visakorpi T Holli K Mäenpää J Endometrial K-ras mutations in postmenopausal breast cancer patients treated with adjuvant tamoxifen or toremifene.Cancer Chemother Pharmacol. 2005; 55: 343-346Crossref PubMed Scopus (24) Google Scholar20Thiede C Creutzig E Illmer T Schaich M Heise V Ehninger G Landt O Rapid and sensitive typing of NPM1 mutations using LNA-mediated PCR clamping.Leukemia. 2006; 20: 1897-1899Crossref PubMed Scopus (34) Google Scholar due to the high limit detection of direct sequencing.10Wang YY Zhou GB Yin T Chen B Shi JY Liang WX Jin XL You JH Yang G Shen ZX Chen J Xiong SM Chen GQ Xu F Liu YW Chen Z Chen SJ AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec.Proc Natl Acad Sci USA. 2005; 102: 1104-1109Crossref PubMed Scopus (268) Google Scholar,13Jones AV Cross NC White HE Green AR Scot LM Rapid identification of JAK2 exon 12 mutations using high resolution melting analysis.Haematologica. 2008; 93: 1560-1564Crossref PubMed Scopus (44) Google Scholar The method is reproducible enough, as it is supported by the practical identity of differential plots of duplicates. The lower limit detection achieved with this method, 1% for exons 8 and 17 KIT mutations, is lower than that accomplished by most of the conventional methods. Furthermore, other relevant advantages of the method are rapidity, low cost, and high-throughput capability, allowing the simultaneous detection of 48 or 192 duplicate samples for the respective 96 or 384 multiwell plates. These characteristics give the HRM setup clear advantages over the methods reported to date,3Paschka P Marcucci G Ruppert AS Mrózek K Chen H Kittles RA Vukosavljevic T Perrotti D Vardiman JW Carroll AJ Kolitz JE Larson RA Bloomfield CD Cancer and Leukemia Group B Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;16).J Clin Oncol. 2006; 24: 3904-3911Crossref PubMed Scopus (545) Google Scholar,5Cairoli R Beghini A Grillo G Nadali G Elice F Ripamonti CB Colapietro P Nichelatti M Pezzetti L Lunghi M Cuneo A Viola A Ferrara F Lazzarino M Rodeghiero F Pizzolo G Larizza L Morra E Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study.Blood. 2006; 107: 3463-3468Crossref PubMed Scopus (302) Google Scholar,9Goemans BF Zwaan CM Miller M Zimmermann M Harlow A Meshinchi S Loonen AH Hählen K Reinhardt D Creutzig U Kaspers GJ Heinrich MC Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia.Leukemia. 2005; 19: 1536-1542Crossref PubMed Scopus (209) Google Scholar,10Wang YY Zhou GB Yin T Chen B Shi JY Liang WX Jin XL You JH Yang G Shen ZX Chen J Xiong SM Chen GQ Xu F Liu YW Chen Z Chen SJ AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and response to Gleevec.Proc Natl Acad Sci USA. 2005; 102: 1104-1109Crossref PubMed Scopus (268) Google Scholar,21Nanri T Matsuno N Kawakita T Suzushima H Kawano F Mitsuya H Asou N Mutations in the receptor tyrosine kinase pathway are associated with clinical outcome in patients with acute myeloblastic leukemia harboring t(8;21)(q22;q22).Leukemia. 2005; 19: 1361-1366Crossref PubMed Scopus (86) Google Scholar including classical real-time PCR methods with labeled probes that are expensive and only detect mutations in the fragment covered by the probes.6Schnittger S Kohl TM Haferlach T Kern W Hiddemann W Spiekermann K Schoch C KIT-D816 mutations in AML1-ETO-positive AML are associated with impaired event-free and overall survival.Blood. 2006; 107: 1791-1799Crossref PubMed Scopus (316) Google Scholar,22Lasa A Carricondo MT Carnicer MJ Perea G Aventín A Nomdedeu JF A new D816 c-KIT gene mutation in refractory AML1-ETO leukemia.Haematologica. 2006; 91: 1283-1284PubMed Google Scholar Previously, Willmore et al23Willmore C Holden JA Zhou L Tripp S Wittwer CT Layfield LJ Detection of c-Kit-activating mutations in gastrointestinal stromal tumors by high-resolution amplicon melting.Am J Clin Pathol. 2004; 122: 206-216Crossref PubMed Scopus (61) Google Scholar used HRM for the screening of KIT gene mutations in gastrointestinal germ cell tumors. However, this HRM assay did not include exon 8, which is a hot spot in AML, and encompasses two steps, initial PCR in LightCycler capillary followed by HRM in an HR-1 instrument with the “drawback problem” of manipulations and time delay. The incidence of KIT mutations in adult patients with CBF AML reported in our study, 18.2% in adults and 7.1% in children, was within the range of 6% to 48% reported for CBF AML.3Paschka P Marcucci G Ruppert AS Mrózek K Chen H Kittles RA Vukosavljevic T Perrotti D Vardiman JW Carroll AJ Kolitz JE Larson RA Bloomfield CD Cancer and Leukemia Group B Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;16).J Clin Oncol. 2006; 24: 3904-3911Crossref PubMed Scopus (545) Google Scholar,5Cairoli R Beghini A Grillo G Nadali G Elice F Ripamonti CB Colapietro P Nichelatti M Pezzetti L Lunghi M Cuneo A Viola A Ferrara F Lazzarino M Rodeghiero F Pizzolo G Larizza L Morra E Prognostic impact of c-KIT mutations in core binding factor leukemias: an Italian retrospective study.Blood. 2006; 107: 3463-3468Crossref PubMed Scopus (302) Google Scholar,6Schnittger S Kohl TM Haferlach T Kern W Hiddemann W Spiekermann K Schoch C KIT-D816 mutations in
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