Sensitive Detection and Quantification of the JAK2V617F Allele by Real-Time PCR
2011; Elsevier BV; Volume: 13; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2011.04.002
ISSN1943-7811
AutoresCornelis J. J. Huijsmans, Jeroen Poodt, Paul H. M. Savelkoul, Mirjam H. A. Hermans,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoA single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617F is somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617F allele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2 was blocked with a peptide nucleic acid oligonucleotide. JAK2V617F patient DNA diluted in JAK2 wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n = 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617F allele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F. A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617F is somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617F allele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2 was blocked with a peptide nucleic acid oligonucleotide. JAK2V617F patient DNA diluted in JAK2 wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n = 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617F allele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F. In 2005, the involvement of the JAK2V617F somatic point mutation in myeloproliferative neoplasms (MPNs) became clear. JAK2V617F is caused by a G-to-T transversion in the Janus kinase 2 (JAK2) gene resulting in a valine-to-phenylalanine amino acid substitution at codon 617.1Kralovics R. Passamonti F. Buser A.S. Teo S.S. Tiedt R. Passweg J.R. Tichelli A. Cazzola M. Skoda R.C. A gain-of-function mutation of JAK2 in myeloproliferative disorders.N Engl J Med. 2005; 352: 1779-1790Crossref PubMed Scopus (2958) Google Scholar, 2James C. Ugo V. Le Couédic J.P. Staerk J. Delhommeau1 F. Lacout C. Garçon L. Raslova H. Berger R. Bennaceur-Griscelli A. Villeval J. Constantinescu S.N. Casadevall N. Vainchenker W. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera.Nature. 2005; 434: 1144-1148Crossref PubMed Scopus (2908) Google Scholar, 3Levine R.L. Wadleigh M. Cools J. Ebert B.L. Wernig G. Huntly B.J. Boggon T.J. Wlodarska I. Clark J.J. Moore S. Adelsperger J. 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Transcriptional responses to polypeptide ligands: the JAK-STAT pathway.Annu Rev Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1649) Google Scholar The autoinhibitory pseudokinase JH2 domain is thought to be by the mutation altered in such a way that the JAK-STAT signaling pathway is constitutively activated, resulting in growth factor–independent cell proliferation.6Steensma D.P. JAK2 V617F in myeloid disorders: molecular diagnostic techniques and their clinical utility: a paper from the 2005 William Beaumont Hospital Symposium on Molecular Pathology.J Mol Diagn. 2006; 8: 397-411Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Since the discovery of JAK2V617F, other mutations in JAK2 and other genes, such as Exon 12, MPL, and TET2, have been described. These mutations occur in a relatively small percentage of patients with MPN.7Tefferi A. Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1.Leukemia. 2010; 24: 1128-1138Crossref PubMed Scopus (435) Google Scholar, 8Scott L.M. Tong W. Levine R.L. Scott M.A. Beer P.A. Stratton M.R. Futreal P.A. Erber W.N. McMullin M.F. Harrison C.N. Warren A.J. Gilliland D.G. Lodish H.F. Green A.R. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis.N Engl J Med. 2007; 356: 459-468Crossref PubMed Scopus (1005) Google Scholar, 9Pikman Y. Lee B.H. Mercher T. McDowell E. Ebert B.L. Gozo M. Cuker A. Wernig G. Moore S. Galinsky I. DeAngelo D.J. Clark J.J. Lee S.J. Golub T.R. Wadleigh M. Gilliland D.G. Levine R.L. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.PLoS Med. 2006; 3: e270Crossref PubMed Scopus (1104) Google Scholar, 10Tefferi A. Pardanani A. Lim K.H. Abdel-Wahab O. Lasho T.L. Patel J. Gangat N. Finke C.M. Schwager S. Mullally A. Li C.Y. Hanson C.A. Mesa R. Bernard O. Delhommeau F. Vainchenker W. Gilliland D.G. Levine R.L. TET2 mutations and their clinical correlates in polycythemia vera, essential thrombocythemia and myelofibrosis.Leukemia. 2009; 23: 905-911Crossref PubMed Scopus (346) Google Scholar JAK2V617F, in contrast, is found in many patients: 65% to 97% of polycythemia vera (PV) cases, 23% to 57% of essential thrombocythemia cases, and 35% to 57% of chronic idiopathic myelofibrosis cases.11Nelson M.E. Steensma D.P. JAK2 V617F in myeloid disorders: what do we know now, and where are we headed?.Leuk Lymphoma. 2006; 47: 177-194Crossref PubMed Scopus (48) Google Scholar, 12Tefferi A. Gilliland D.G. The JAK2V617F tyrosine kinase mutation in myeloproliferative disorders: status report and immediate implications for disease classification and diagnosis.Mayo Clin Proc. 2005; 80: 947-958Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar The diagnostic value of JAK2 mutational analysis in MPN is now well established and endorsed in the classification of hematologic malignancies by the World Health Organization.13Vardiman J.W. Thiele J. Arber D.A. Brunning R.D. Borowitz M.J. Porwit A. Harris N.L. Le Beau M.M. Hellström-Lindberg E. Tefferi A. Bloomfield C.D. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes.Blood. 2009; 11: 937-951Crossref Scopus (3464) Google Scholar Patients with PV often carry a JAK2V617F homozygous burden, in contrast to those with essential thrombocythemia, in whom homozygosity is rare.14Scott L.M. Scott M.A. Campbell P.J. Green A.R. Progenitors homozygous for the V617F mutation occur in most patients with polycythemia vera, but not essential thrombocythemia.Blood. 2006; 108: 2435-2437Crossref PubMed Scopus (228) Google Scholar This finding was also recapitulated in a large multicenter study: PV (n = 323: 67.8% heterozygous and 32.2% homozygous) and essential thrombocythemia (n = 639: 40.2% wild-type, 57.6% heterozygous, and 2.2% homozygous).15Vannucchi A.M. Antonioli E. Guglielmelli P. Rambaldi A. Barosi G. Marchioli R. Marfisi R.M. Finazzi G. Guerini V. Fabris F. Randi M.L. De Stefano V. Caberlon S. Tafuri A. Ruggeri M. Specchia G. Liso V. Rossi E. Pogliani E. Gugliotta L. Bosi A. Barbui T. Clinical profile of homozygous JAK2 617V>F mutation in patients with polycythemia vera or essential thrombocythemia.Blood. 2007; 110: 840-846Crossref PubMed Scopus (372) Google Scholar It is, therefore, more common for patients with PV to have a mutant allele burden >50%. Although the clinical value of the JAK2V617F allele burden is not yet fully understood, a correlation between disease phenotype and the proportion of mutant alleles has been postulated.15Vannucchi A.M. Antonioli E. Guglielmelli P. Rambaldi A. Barosi G. Marchioli R. Marfisi R.M. Finazzi G. Guerini V. Fabris F. Randi M.L. De Stefano V. Caberlon S. Tafuri A. Ruggeri M. Specchia G. Liso V. Rossi E. Pogliani E. Gugliotta L. Bosi A. Barbui T. Clinical profile of homozygous JAK2 617V>F mutation in patients with polycythemia vera or essential thrombocythemia.Blood. 2007; 110: 840-846Crossref PubMed Scopus (372) Google Scholar, 16Passamonti F. Rumi E. Clinical relevance of JAK2 (V617F) mutant allele burden.Haematologica. 2009; 94: 7-10Crossref PubMed Scopus (64) Google Scholar Splenomegaly, vascular events and pruritus in PV and splenomegaly, arterial thrombosis, and microvessel disease in essential thrombocythemia have been clinically correlated to mutant allele burdens.17Vannucchi A.M. Antonioli E. Guglielmelli P. Longo G. Pancrazzi A. Ponziani V. Bogani C. Ferrini P.R. Rambaldi A. Guerini V. Bosi A. Barbui T. MPD Research ConsortiumProspective identification of high-risk polycythemia vera patients based on JAK2(V617F) allele burden.Leukemia. 2007; 21: 1952-1959Crossref PubMed Scopus (265) Google Scholar, 18Antonioli E. Guglielmelli P. Poli G. Bogani C. Pancrazzi A. Longo G. Ponziani V. Tozzi L. Pieri L. Santini V. Bosi A. Vannucchi A.M. Myeloproliferative Disorders Research Consortium (MPD-RC)Influence of JAK2V617F allele burden on phenotype in essential thrombocythemia.Haematologica. 2008; 93: 41-48Crossref PubMed Scopus (133) Google Scholar Patients in the lowest quartile of mutant allele burden in idiopathic myelofibrosis had, overall, significantly shortened overall and leukemia-free survival compared with those with higher allele burdens and those negative for JAK2V617F.19Tefferi A. Lasho T.L. Schwager S.M. Strand J.S. Elliott M. Mesa R. Li C.Y. Wadleigh M. Lee S.J. Gilliland D.G. The clinical phenotype of wild-type, heterozygous, and homozygous JAK2V617F in polycythemia vera.Cancer. 2006; 106: 631-635Crossref PubMed Scopus (194) Google Scholar In addition, the amount of mutant allele burden in correlation to therapy may be of increasing interest in future diagnostic and predictive tests.20Tefferi A. Mutational analysis in BCR-ABL-negative classic myeloproliferative neoplasms: impact on prognosis and therapeutic choices.Leuk Lymphoma. 2010; 51: 576-582Crossref PubMed Scopus (11) Google Scholar With the discovery of JAK2V617F, the detection of this mutation became routine procedure in many laboratories. Since 2005, many assays to detect, and sometimes quantify, JAK2V617F have been described, evaluated, and compared.4Baxter E.J. Scott L.M. Campbell P.J. East C. Fourouclas N. Swanton S. Vassiliou G.S. Bench A.J. Boyd E.M. Curtin N. Scott M.A. Erber W.N. Green A.R. 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Automated JAK2V617F quantification using a magnetic filtration system and sequence-specific primer-single molecule fluorescence detection.Cancer Genet Cytogenet. 2007; 179: 19-24Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar Today, numerous techniques are used: PCR–restriction fragment length polymorphism (RFLP), direct sequencing, pyrosequencing, allele-specific PCR/amplification refractory mutation system, allelic discrimination, real-time PCR, high-resolution melting curve analysis, PCR, denaturing high-pressure liquid chromatography, PCR–matrix-assisted laser desorption/ionization–time of flight (PCR-MALDI-TOF) mass spectrometry, and sequence-specific primer–single molecule fluorescence detection. The techniques differ in a variety of characteristics, such as sensitivity (ranging from 0.01% to 20%, mutant allele diluted in wild-type allele), the potential for high-throughput diagnostics, and equipment required. An overview of currently available assays is presented in Table 1.Table 1Overview and Some Characteristics of Currently Available JAK2V617F Detection (and Quantification) TechniquesTechniqueSensitivity (%)AdvantagesDisadvantagesReferencesPCR-RFLP1–20No special equipment required, inexpensiveRisk of false positives due to incomplete digestion, labor intensive, post-PCR processing required4Baxter E.J. Scott L.M. Campbell P.J. East C. Fourouclas N. Swanton S. Vassiliou G.S. Bench A.J. Boyd E.M. Curtin N. Scott M.A. Erber W.N. Green A.R. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders.Lancet. 2005; 365: 1054-1061Abstract Full Text Full Text PDF PubMed Scopus (2296) Google Scholar, 6Steensma D.P. JAK2 V617F in myeloid disorders: molecular diagnostic techniques and their clinical utility: a paper from the 2005 William Beaumont Hospital Symposium on Molecular Pathology.J Mol Diagn. 2006; 8: 397-411Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 21Cankovic M. Whiteley L. Hawley R.C. Zarbo R.J. Chitale D. Clinical performance of JAK2 V617F mutation detection assays in a molecular diagnostics laboratory: evaluation of screening and quantitation methods.Am J Clin Pathol. 2009; 132: 713-721Crossref PubMed Scopus (34) Google Scholar, 22Frantz C. Sekora D.M. Henley D.C. Huang C.K. Pan Q. Quigley N.B. Gorman E. Hubbard R.A. Mirza I. Comparative evaluation of three JAK2V617F mutation detection methods.Am J Clin Pathol. 2007; 128: 865-874Crossref PubMed Scopus (21) Google Scholar, 23Kannim S. Thongnoppakhun W. Auewarakul C.U. Two-round allele specific-polymerase chain reaction: a simple and highly sensitive method for JAK2V617F mutation detection.Clin Chim Acta. 2009; 401: 148-151Crossref PubMed Scopus (22) Google Scholar, 24Kremer M. Horn T. Koch I. Dechow T. Gattenloehner S. Pfeiffer W. Quintanilla-Martínez L. Fend F. Quantitation of the JAK2V617F mutation in microdissected bone marrow trephines: equal mutational load in myeloid lineages and rare involvement of lymphoid cells.Am J Surg Pathol. 2008; 32: 928-935Crossref PubMed Scopus (11) Google Scholar, 25Lay M. Mariappan R. Gotlib J. Dietz L. Sebastian S. Schrijver I. Zehnder J.L. Detection of the JAK2 V617F mutation by LightCycler PCR and probe dissociation analysis.J Mol Diagn. 2006; 8: 330-334Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 26Shammaa D. Bazarbachi A. Halas H. Greige L. Mahfouz R. JAK2 V617F mutation detection: laboratory comparison of two kits using RFLP and qPCR.Genet Test Mol Biomarkers. 2010; 14: 13-15Crossref PubMed Scopus (4) Google ScholarDirect sequencing5–20Simultaneous detection of other mutationsLabor intensive, sequencing equipment required, post-PCR processing required4Baxter E.J. Scott L.M. Campbell P.J. East C. Fourouclas N. Swanton S. Vassiliou G.S. Bench A.J. Boyd E.M. Curtin N. Scott M.A. Erber W.N. Green A.R. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders.Lancet. 2005; 365: 1054-1061Abstract Full Text Full Text PDF PubMed Scopus (2296) Google Scholar, 6Steensma D.P. JAK2 V617F in myeloid disorders: molecular diagnostic techniques and their clinical utility: a paper from the 2005 William Beaumont Hospital Symposium on Molecular Pathology.J Mol Diagn. 2006; 8: 397-411Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 22Frantz C. Sekora D.M. Henley D.C. Huang C.K. Pan Q. Quigley N.B. 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