Allele-Specific Polymerase Chain Reaction for the Imatinib-Resistant KIT D816V and D816F Mutations in Mastocytosis and Acute Myelogenous Leukemia
2006; Elsevier BV; Volume: 8; Issue: 5 Linguagem: Inglês
10.2353/jmoldx.2006.060089
ISSN1943-7811
AutoresChristopher L. Corless, Patina M. Harrell, Mario E. Lacouture, Troy Bainbridge, Claudia Le, Ken Gatter, Clifton R. White, Scott R. Granter, Michael C. Heinrich,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoOncogenic mutations of the receptor tyrosine kinase KIT contribute to the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis (SM), and some cases of acute myelogenous leukemia (AML). The D816V substitution in the activation loop of KIT results in relative resistance to the kinase inhibitor imatinib (Gleevec). Because this mutation occurs in 80 to 95% of adult SM, its detection has diagnostic and predictive significance. Unfortunately, the fraction of mutation-positive cells in clinical SM samples is often below the 20 to 30% threshold needed for detection by direct DNA sequencing. We have developed an allele-specific polymerase chain reaction assay using a mutation-specific primer combined with a wild-type blocking oligonucleotide that amplifies D816V at the level of 1% mutant allele in DNA extracted from formalin-fixed, paraffin-embedded tissue. There were no amplifications among 64 KIT wild-type tumors and cell lines, whereas all D816V-mutant samples (eight AML and 11 mast cell disease) were positive. Other D816 substitutions associated with resistance to imatinib in vitro are rare in SM. Among these D816F was detectable with the assay whereas D816H, D816Y, and D816G did not amplify. Nine biopsies (bone marrow, skin, or colon) with suspected SM were negative by denaturing high performance liquid chromatography and/or DNA sequencing but positive by allele-specific polymerase chain reaction. Thus, the assay may be useful in confirming the diagnosis of SM. Oncogenic mutations of the receptor tyrosine kinase KIT contribute to the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis (SM), and some cases of acute myelogenous leukemia (AML). The D816V substitution in the activation loop of KIT results in relative resistance to the kinase inhibitor imatinib (Gleevec). Because this mutation occurs in 80 to 95% of adult SM, its detection has diagnostic and predictive significance. Unfortunately, the fraction of mutation-positive cells in clinical SM samples is often below the 20 to 30% threshold needed for detection by direct DNA sequencing. We have developed an allele-specific polymerase chain reaction assay using a mutation-specific primer combined with a wild-type blocking oligonucleotide that amplifies D816V at the level of 1% mutant allele in DNA extracted from formalin-fixed, paraffin-embedded tissue. There were no amplifications among 64 KIT wild-type tumors and cell lines, whereas all D816V-mutant samples (eight AML and 11 mast cell disease) were positive. Other D816 substitutions associated with resistance to imatinib in vitro are rare in SM. Among these D816F was detectable with the assay whereas D816H, D816Y, and D816G did not amplify. Nine biopsies (bone marrow, skin, or colon) with suspected SM were negative by denaturing high performance liquid chromatography and/or DNA sequencing but positive by allele-specific polymerase chain reaction. Thus, the assay may be useful in confirming the diagnosis of SM. The receptor tyrosine kinase KIT plays an important role in the development of bone marrow stem cells, mast cells, primary germ cells, and the interstitial (Cajal) cells of the gastrointestinal tract.1Heinrich MC Blanke CD Druker BJ Corless CL Inhibition of KIT tyrosine kinase activity: a novel molecular approach to the treatment of KIT positive malignancies.J Clin Oncol. 2002; 20: 1692-1703Crossref PubMed Scopus (575) Google Scholar Correspondingly, gain of function mutations in the KIT gene are found in myeloproliferative disorders, including subtypes of mastocytosis and in seminomas and gastrointestinal stromal tumors (GISTs).1Heinrich MC Blanke CD Druker BJ Corless CL Inhibition of KIT tyrosine kinase activity: a novel molecular approach to the treatment of KIT positive malignancies.J Clin Oncol. 2002; 20: 1692-1703Crossref PubMed Scopus (575) Google Scholar,2Corless CL Fletcher JA Heinrich MC Biology of gastrointestinal stromal tumors.J Clin Oncol. 2004; 22: 3813-3825Crossref PubMed Scopus (997) Google Scholar With the introduction of KIT kinase inhibitors such as imatinib (Gleevec) into clinical practice, there is increasing recognition of the critical relationship between KIT gene mutations and the response to treatment. For example, the 60 to 70% of GISTs that harbor a mutation in the juxtamembrane domain of KIT (exon 11) are more responsive to imatinib therapy than are GISTs with no KIT mutation.3Debiec-Rychter M Dumez H Judson I Wasag B Verweij J Brown M Dimitrijevic S Sciot R Stul M Vranck H Scurr M Hagemeijer A van Glabbeke M Van Oosterom AT Use of c-KIT/PDGFRA mutational analysis to predict the clinical response to imatinib in patients with advanced gastrointestinal stromal tumours entered on phase I and II studies of the EORTC Soft Tissue and Bone Sarcoma Group.Eur J Cancer. 2004; 40: 689-695Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar,4Heinrich MC Corless CL Demetri GD Blanke CD von Mehren M Joensuu H McGreevey LS Chen CJ Van den Abbeele AD Druker BJ Kiese B Eisenberg B Roberts PJ Singer S Fletcher CD Silberman S Dimitrijevic S Fletcher JA Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor.J Clin Oncol. 2003; 21: 4342-4349Crossref PubMed Scopus (1934) Google Scholar Interestingly, these exon 11-mutant tumors may become resistant to imatinib by acquiring a secondary KIT mutation in the ATP-binding pocket (exon 13) or activation loop domain (exon 17).5Antonescu CR Besmer P Guo T Arkun K Hom G Koryotowski B Leversha MA Jeffrey PD Desantis D Singer S Brennan MF Maki RG DeMatteo RP Acquired resistance to imatinib in gastrointestinal stromal tumor occurs through secondary gene mutation.Clin Cancer Res. 2005; 11: 4182-4190Crossref PubMed Scopus (678) Google Scholar6Debiec-Rychter M van Oosterom A Marynen P Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants.Gastroenterology. 2005; 128: 270-279Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar7Wakai T Kanda T Hirota S Ohashi A Shirai Y Hatakeyama K Late resistance to imatinib therapy in a metastatic gastrointestinal stromal tumour is associated with a second KIT mutation.Br J Cancer. 2004; 90: 2059-2061Crossref PubMed Scopus (88) Google Scholar8Wardelmann E Thomas N Merkelbach-Bruse S Pauls K Speidel N Buttner R Bihl H Leutner CC Heinicke T Hohenberger P Acquired resistance to imatinib in gastrointestinal stromal tumours caused by multiple KIT mutations.Lancet Oncol. 2005; 6: 249-251Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar Exon 17 is of particular significance in this regard because primary mutations in this domain are found in systemic mastocytosis (SM) and acute myelogenous leukemia (AML), and there is clinical interest in the use of kinase inhibitors like imatinib to treat these diseases.The first KIT gene mutation identified in human cells was the substitution of valine for aspartic acid at codon 816 (D816V; A81402T based on GenBank U63834; KIT cDNA A2468T) in a mast cell leukemia cell line.9Furitsu T Tsujimura T Tono T Ikeda H Kitayama H Koshimizu U Sugahara H Butterfield JH Ashman LK Kanayama Y Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukemia cell line causing ligand-independent activation of c-kit product.J Clin Invest. 1993; 92: 1736-1744Crossref PubMed Scopus (732) Google Scholar The resulting mutant form of KIT has a constitutively activated kinase, signaling from which appears to be essential for the survival and proliferation of mast cell precursors in the marrow. Studies from a number of groups have established that substitutions at codon 816 are common in systemic mast cell disease, and such mutations are defined by the World Health Organization as one of the minor criteria for the diagnosis of SM.10Akin C Metcalfe DD Systemic mastocytosis.Annu Rev Med. 2004; 55: 419-432Crossref PubMed Scopus (195) Google Scholar11Longley BJ Metcalfe DD A proposed classification of mastocytosis incorporating molecular genetics.Hematol Oncol Clin North Am. 2000; 13: 697-701Abstract Full Text Full Text PDF Scopus (45) Google Scholar12Metcalfe DD Akin C Mastocytosis: molecular mechanisms and clinical disease heterogeneity.Leuk Res. 2001; 25: 577-582Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar13Tefferi A Pardanani A Clinical, genetic, and therapeutic insights into systemic mast cell disease.Curr Opin Hematol. 2004; 11: 58-64Crossref PubMed Scopus (80) Google Scholar14Valent P Akin C Sperr W Mayerhofer M Fodinger M Fritsche-Polanz R Sotlar K Escribano L Arock M Horny HP Metcalfe D Mastocytosis: pathology, genetics, and current options for therapy.Leuk Lymphoma. 2005; 46: 35-48Crossref PubMed Scopus (184) Google Scholar15Garcia-Montero AC, Jara-Acevedo M, Teodosio C, Sanchez ML, Nunez R, Prados A, Aldanondo I, Sanchez L, Dominguez M, Botana LM, Sanchez-Jimenez F, Sotlar K, Almeida J, Escribano L, Orfao A: KIT mutation in mast cells and other bone marrow haematopoietic cell lineages in systemic mast cell disorders. A prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients. Blood epub June 1, 2006Google Scholar The classification for SM includes indolent SM, aggressive SM, mast cell leukemia, and SM with associated hematological nonmast cell lineage disease (SM-AHNMD), which includes variants with associated myelodysplastic syndrome (SM-MDS), myeloproliferative disease (SM-MPD), and acute myelogenous leukemia (SM-AML).14Valent P Akin C Sperr W Mayerhofer M Fodinger M Fritsche-Polanz R Sotlar K Escribano L Arock M Horny HP Metcalfe D Mastocytosis: pathology, genetics, and current options for therapy.Leuk Lymphoma. 2005; 46: 35-48Crossref PubMed Scopus (184) Google Scholar,16Valent P Akin C Sperr WR Horny HP Metcalfe DD Mast cell proliferative disorders: current view on variants recognized by the World Health Organization.Hematol Oncol Clin North Am. 2003; 17: 1227-1241Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar KIT gene mutations have been identified in all of these SM subtypes, being detectable in samples of skin, bone marrow, gut, spleen, and blood, albeit to varying degrees.14Valent P Akin C Sperr W Mayerhofer M Fodinger M Fritsche-Polanz R Sotlar K Escribano L Arock M Horny HP Metcalfe D Mastocytosis: pathology, genetics, and current options for therapy.Leuk Lymphoma. 2005; 46: 35-48Crossref PubMed Scopus (184) Google ScholarAmong the KIT codon 816 substitutions reported, D816V is by far the most common, representing more than 90% of mutations in adult SM patients in a number of series.17Akin C Kirshenbaum AS Semere T Worobec AS Scott LM Metcalfe DD Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis.Exp Hematol. 2000; 28: 140-147Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar18Buttner C Henz BM Welker P Sepp NT Grabbe J Identification of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a possible explanation for divergent clinical behavior.J Invest Dermatol. 1998; 111: 1227-1231Crossref PubMed Scopus (192) Google Scholar19Fritsche-Polanz R Jordan JH Feix A Sperr WR Sunder-Plassmann G Valent P Fodinger M Mutation analysis of C-KIT in patients with myelodysplastic syndromes without mastocytosis and cases of systemic mastocytosis.Br J Haematol. 2001; 113: 357-364Crossref PubMed Scopus (134) Google Scholar20Longley BJJ Metcalfe DD Tharp M Wang X Tyrrell L Lu SZ Heitjan D Ma Y Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.Proc Natl Acad Sci USA. 1999; 96: 1609-1614Crossref PubMed Scopus (494) Google Scholar21Nagata H Worobec AS Oh CK Chowdhury BA Tannenbaum S Suzuki Y Metcalfe DD Identification of a point mutation in the catalytic domain of the protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder.Proc Natl Acad Sci USA. 1995; 92: 10560-10564Crossref PubMed Scopus (792) Google Scholar22Yanagihori H Oyama N Nakamura K Kaneko F c-kit Mutations in patients with childhood-onset mastocytosis and genotype-phenotype correlation.J Mol Diagn. 2005; 7: 252-257Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar In the largest series to date, Garcia-Montero and colleagues15Garcia-Montero AC, Jara-Acevedo M, Teodosio C, Sanchez ML, Nunez R, Prados A, Aldanondo I, Sanchez L, Dominguez M, Botana LM, Sanchez-Jimenez F, Sotlar K, Almeida J, Escribano L, Orfao A: KIT mutation in mast cells and other bone marrow haematopoietic cell lineages in systemic mast cell disorders. A prospective study of the Spanish Network on Mastocytosis (REMA) in a series of 113 patients. Blood epub June 1, 2006Google Scholar observed KIT mutations in 93% of 113 cases, and nearly all of these (97%) were D816V. Other, more rare KIT mutations that have been reported in SM include alterations in exon 17 (D815K, D816F, D816H, D816Y, D820G, E839K), exon 11 (V560G), and exon 10 (K509I, F522C, V530I, A533D). Among these, D816F may be relatively more common in pediatric patients.22Yanagihori H Oyama N Nakamura K Kaneko F c-kit Mutations in patients with childhood-onset mastocytosis and genotype-phenotype correlation.J Mol Diagn. 2005; 7: 252-257Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar,23Sotlar K Escribano L Landt O Mohrle S Herrero S Torrelo A Lass U Horny HP Bultmann 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 (184) Google Scholar Interestingly, in SM with excess eosinophils (SM-EOS, hypereosinophilic syndrome), the fusion oncogene FIP1L1-PDGFRA is the most commonly detected molecular alteration.24Cools J DeAngelo DJ Gotlib J Stover EH Legare RD Cortes J Kutok J Clark J Galinsky I Griffin JD Cross NC Tefferi A Malone J Alam R Schrier SL Schmid J Rose M Vandenberghe P Verhoef G Boogaerts M Wlodarska I Kantarjian H Marynen P Coutre SE Stone R Gilliland DG A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome.N Engl J Med. 2003; 348: 1201-1214Crossref PubMed Scopus (1486) Google Scholar25Pardanani A Brockman SR Paternoster SF Flynn HC Ketterling RP Lasho TL Ho CL Li CY Dewald GW Tefferi A FIP1L1-PDGFRA fusion: prevalence and clinicopathologic correlates in 89 consecutive patients with moderate to severe eosinophilia.Blood. 2004; 104: 3038-3045Crossref PubMed Scopus (267) Google Scholar26Pardanani A Tefferi A Imatinib therapy for hypereosinophilic syndrome and eosinophilia-associated myeloproliferative disorders.Leuk Res. 2004; 28: 47-52Abstract Full Text Full Text PDF Scopus (24) Google ScholarDetection of KIT D816V is significant not only because it can help confirm a suspected diagnosis of SM, but because this mutant form of KIT is fully resistant to inhibition by imatinib in vitro,4Heinrich MC Corless CL Demetri GD Blanke CD von Mehren M Joensuu H McGreevey LS Chen CJ Van den Abbeele AD Druker BJ Kiese B Eisenberg B Roberts PJ Singer S Fletcher CD Silberman S Dimitrijevic S Fletcher JA Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor.J Clin Oncol. 2003; 21: 4342-4349Crossref PubMed Scopus (1934) Google Scholar,27Frost MJ Ferrao PT Hughes TP Ashman LK Juxtamembrane mutant V560GKit is more sensitive to imatinib (STI571) compared with wild-type c-kit whereas the kinase domain mutant D816VKit is resistant.Mol Cancer Ther. 2002; 1: 1115-1124PubMed Google Scholar28Ma Y Zeng S Metcalfe DD Akin C Dimitrijevic S Butterfield JH McMahon G Longley BJ The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations.Blood. 2002; 99: 1741-1744Crossref PubMed Scopus (407) Google Scholar29Zermati Y De Sepulveda P Feger F Letard S Kersual J Casteran N Gorochov G Dy M Ribadeau DA Dorgham K Parizot C Bieche Y Vidaud M Lortholary O Arock M Hermine O Dubreuil P Effect of tyrosine kinase inhibitor STI571 on the kinase activity of wild-type and various mutated c-kit receptors found in mast cell neoplasms.Oncogene. 2003; 22: 660-664Crossref PubMed Scopus (163) Google Scholar and clinical reports suggest that this drug is not very effective in the treatment of D816V-positive disease.30Hennessy B Giles F Cortes J O'Brien S Ferrajoli A Ossa G Garcia-Manero G Faderl S Kantarjian H Verstovsek S Management of patients with systemic mastocytosis: review of M. D. Anderson Cancer Center experience.Am J Hematol. 2004; 77: 209-214Crossref PubMed Scopus (47) Google Scholar31Musto P Falcone A Sanpaolo G Bodenizza C Carella AM Inefficacy of imatinib-mesylate in sporadic, aggressive systemic mastocytosis.Leuk Res. 2004; 28: 421-422Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar32Pardanani A Elliott M Reeder T Li CY Baxter EJ Cross NC Tefferi A Imatinib for systemic mast-cell disease.Lancet. 2003; 362: 535-536Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar33Worobec AS Semere T Nagata H Metcalfe DD Clinical correlates of the presence of the Asp816Val c-kit mutation in the peripheral blood mononuclear cells of patients with mastocytosis.Cancer. 1998; 83: 2120-2129Crossref PubMed Scopus (120) Google Scholar34Droogendijk HJ Kluin-Nelemans HJ Van Doormaal JJ Oranje AP van de Loosdrecht AA van Daele PL Imatinib mesylate in the treatment of systemic mastocytosis: a phase II trial.Cancer. 2006; 107: 345-351Crossref PubMed Scopus (123) Google Scholar D816V is also observed in cases of AML, particularly in tumors with core binding factor translocations,35Beghini A Ripamonti CB Cairoli R Cazzaniga G Colapietro P Elice F Nadali G Grillo G Haas OA Biondi A Morra E Larizza L KIT activating mutations: incidence in adult and pediatric acute myeloid leukemia, and identification of an internal tandem duplication.Haematologica. 2004; 89: 920-925PubMed Google Scholar,36Goemans BF Zwaan C Miller M Zimmermann M Harlow A Meshinchi S Loonen AH Hahlen 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 (206) Google Scholar and there is evidence that D816V confers resistance to imatinib treatment in this setting as well.37Cairoli R Beghini A Morello E Grillo G Montillo M Larizza L Morra E Imatinib mesylate in the treatment of core binding factor leukemias with KIT mutations. A report of three cases.Leuk Res. 2005; 29: 397-400Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar For these reasons, the KIT D816V mutation is of particular clinical interest.Determining the KIT gene mutation status in cases of AML is generally straightforward because tumor cells typically dominate blood, bone marrow aspirate, and bone marrow biopsy samples. In contrast, mutation analysis in mastocytosis presents a greater challenge. The degree of mast cell infiltration in affected organs varies widely and may represent as little as 1% of the cell population in a given biopsy. In recent studies of GISTs, we have used denaturing high-performance liquid chromatography (HPLC) to screen KIT gene amplicons for mutations.4Heinrich MC Corless CL Demetri GD Blanke CD von Mehren M Joensuu H McGreevey LS Chen CJ Van den Abbeele AD Druker BJ Kiese B Eisenberg B Roberts PJ Singer S Fletcher CD Silberman S Dimitrijevic S Fletcher JA Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor.J Clin Oncol. 2003; 21: 4342-4349Crossref PubMed Scopus (1934) Google Scholar,38Corless CL McGreevey L Haley A Town A Heinrich MC KIT mutations are common in incidental gastrointestinal stromal tumors one centimeter or less in size.Am J Pathol. 2002; 160: 1567-1572Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar With a sensitivity of ∼10 to 20% mutant allele, HPLC works well in the analysis of DNA from the highly cellular samples of these sarcomas. However, analysis of biopsies from patients with possible SM is problematic using this methodology because the number of mast cells is often quite low (<10% of cells) and the probability of a false negative result is unacceptably high. In response to growing clinical demand for KIT mutation screening in patients with suspected SM, we looked at published methods that have been used to detect KIT mutations in this patient population. These include cDNA amplification and sequencing, analyses of genomic DNA by direct sequencing, restriction fragment length polymorphism or single strand conformation polymorphism, and real-time polymerase chain reaction (PCR) using peptide-nucleic acid probes.18Buttner C Henz BM Welker P Sepp NT Grabbe J Identification of activating c-kit mutations in adult-, but not in childhood-onset indolent mastocytosis: a possible explanation for divergent clinical behavior.J Invest Dermatol. 1998; 111: 1227-1231Crossref PubMed Scopus (192) Google Scholar19Fritsche-Polanz R Jordan JH Feix A Sperr WR Sunder-Plassmann G Valent P Fodinger M Mutation analysis of C-KIT in patients with myelodysplastic syndromes without mastocytosis and cases of systemic mastocytosis.Br J Haematol. 2001; 113: 357-364Crossref PubMed Scopus (134) Google Scholar20Longley BJJ Metcalfe DD Tharp M Wang X Tyrrell L Lu SZ Heitjan D Ma Y Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.Proc Natl Acad Sci USA. 1999; 96: 1609-1614Crossref PubMed Scopus (494) Google Scholar,22Yanagihori H Oyama N Nakamura K Kaneko F c-kit Mutations in patients with childhood-onset mastocytosis and genotype-phenotype correlation.J Mol Diagn. 2005; 7: 252-257Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar,23Sotlar K Escribano L Landt O Mohrle S Herrero S Torrelo A Lass U Horny HP Bultmann 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 (184) Google Scholar,33Worobec AS Semere T Nagata H Metcalfe DD Clinical correlates of the presence of the Asp816Val c-kit mutation in the peripheral blood mononuclear cells of patients with mastocytosis.Cancer. 1998; 83: 2120-2129Crossref PubMed Scopus (120) Google Scholar Because most clinical samples are formalin-fixed and paraffin-embedded, a cDNA-based approach would be technically challenging. Restriction fragment length polymorphism and single strand conformation polymorphism are somewhat labor intensive and have limited sensitivities, and our attempts to adopt the peptide-nucleic acid probe method described by Sotlar and colleagues23Sotlar K Escribano L Landt O Mohrle S Herrero S Torrelo A Lass U Horny HP Bultmann 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 (184) Google Scholar were unsuccessful. Therefore, we developed a new allele-specific PCR (AS-PCR) method that is modeled on a RAS gene assay reported by McKinzie and Parsons.39McKinzie PB Parsons BL Detection of rare K-ras codon 12 mutations using allele-specific competitive blocker PCR.Mutat Res. 2002; 517: 209-220Crossref PubMed Scopus (43) Google Scholar This AS-PCR assay is based on a combination of a mutation-directed primer and a wild-type blocking oligonucleotide and can reproducibly detect either KIT D816V or D816F that is present at low levels in DNA extracted from paraffin-embedded tissue.Materials and MethodsTumor Samples and Cell LinesTumor samples were obtained from the Tissue Bank of the Oregon Health and Science University Cancer Institute, from the Oregon Health and Science University Departments of Pathology and Dermatology, the University of Chicago Section of Dermatology, and the Department of Pathology of Brigham and Women's Hospital. The use of these tissues in the study was performed in accordance with the regulations of the institutional review boards of these institutions. The human HMC-1.2 mast cell line, which harbors both V560D and D816V mutations of the KIT gene, was kindly provided by Dr. C. Akin (Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD).28Ma Y Zeng S Metcalfe DD Akin C Dimitrijevic S Butterfield JH McMahon G Longley BJ The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations.Blood. 2002; 99: 1741-1744Crossref PubMed Scopus (407) Google Scholar LoVo cells (a colon cancer cell line that is wild type for KIT) were obtained from the American Type Culture Collection, Rockville, MD (http://www.atcc.org). In a previous study we generated stably transfected clones of Ba/F3 cells (murine interleukin 3-dependent hematopoietic pro-B cells) expressing wild-type, D816V, D816F, or D816Y mutant forms of human KIT cDNA.40Schittenhelm MM Shiraga S Schroeder A Corbin AS Griffith D Lee FY Bokemeyer C Deininger MW Druker BJ Heinrich MC Dasatinib (BMS-354825), a dual SRC/ABL kinase inhibitor, inhibits the kinase activity of wild-type, juxtamembrane, and activation loop mutant KIT isoforms associated with human malignancies.Cancer Res. 2006; 66: 473-481Crossref PubMed Scopus (423) Google Scholar DNA extracted from these Ba/F3 subclones was used in validation experiments.DNA PreparationDNA was extracted from HMC-1.2, LoVo, and Ba/F3 cells with the QIAmp DNA mini kit (no. 51306; Qiagen, Valencia, CA). The same kit was used to extract genomic DNA from blood and bone marrow aspirates, as well as from formalin-fixed tissue (unembedded), and formalin-fixed, paraffin-embedded tissue samples, in accordance with the manufacturer's recommendations.Standard PCRKIT exon 17 amplicons were generated from genomic DNA as previously described.41Kemmer K Corless CL Fletcher JA McGreevey L Haley A Griffith D Cummings OW Wait C Town A Heinrich MC KIT mutations are common in testicular seminomas.Am J Pathol. 2004; 164: 305-313Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar For the standard (control) PCR reaction, the forward primer was 5′-TGTATTCACAGAGACTTGGC-3′, and the reverse primer was 5′-TAATGTTCAGCATACCATGCAA-3′. This reverse primer matches sequences in KIT intron 17 and was also used in the allele-specific PCR. To amplify KIT cDNA sequences from Ba/F3 subclones, the same forward primer was matched with the reverse primer 5′-GCTCCCAAAGAAAAATCCCATAGG-3′.Allele-Specific PCRAllele-specific PCR reactions were performed using 200 ng of DNA and a master mix based on the Expand High Fidelity Polymerase kit (no. 11759078001; Roche, Indianapolis, IN) with 1.4 U of polymerase, 160 μmol/L dNTP (Stratagene, Cedar Creek, TX), 400 nmol/L mutation-specific primer (Figure 1), 200 nmol/L blocking oligonucleotide (Figure 1), and 800 nmol/L reverse primer (5′-TAATGTTCAGCATACCATGCAA-3′). The cycling conditions were as follows: 95°C for 1 minute, followed by 45 cycles of 94°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute, and a final 7-minute incubation at 73°C.HPLC and DNA SequencingPCR products were routinely analyzed on a denaturing HPLC system (WAVE; Transgenomic, Inc., Omaha, NE), as previously described.4Heinrich MC Corless CL Demetri GD Blanke CD von Mehren M Joensuu H McGreevey LS Chen CJ Van den Abbeele AD Druker BJ Kiese B Eisenberg B Roberts PJ Singer S Fletcher CD Silberman S Dimitrijevic S Fletcher JA Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor.J Clin Oncol. 2003; 21: 4342-4349Crossref PubMed Scopus (1934) Google Scholar,38Corless CL McGreevey L Haley A Town A Heinrich MC KIT mutations are common in incidental gastrointestinal stromal tumors one centimeter or less in size.Am J Pathol. 2002; 160: 1567-1572Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar This system allows the detection of DNA heteroduplexes that differ by as little as one nucleotide in 400 bp and is sensitive in the range of 15 to 20% mutant allele. DNA sequencing was performed using the BIGDye terminator kit (Applied Biosystems, Foster City, CA) and analyzed on an ABI 310 capillary sequencer.4Heinrich MC Corless CL Demetri GD Blanke CD von Mehren M Joensuu H McGreevey LS Chen CJ Van den Abbeele AD Druker BJ Kiese B Eisenberg B Roberts PJ Singer S Fletcher CD Silberman S Dimitrijevic S Fletcher JA Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor.J Clin Oncol. 2003; 21: 4342-4349Crossref PubMed Scopus (1934) Google ScholarTopo SubcloningTo look for low-abundance KIT exon 17 mutations, amplicons were cloned into Topo plasmids and sequenced individually, as previously described.42Deininger MW McGreevey L Willis S Bainbridge TM Druker BJ Heinrich MC Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography.Leukemia. 2004; 18: 864-871Crossref PubMed Scopus (59) Google ScholarResultsAssay DesignOur assay is modeled on the approach recently described by McKinzie and Parsons39McKinzie PB Parsons BL Detection of rare K-ras codon 12 mutations using allele-specific competitive blocker PCR.Mutat Res
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