Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2^V617F Mutation

Abstract

Precise quantification of the JAK2^V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2^V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988).
In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2^V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations.

Keywords

myeloproliferative neoplasms / JAK2 V617F mutation / ddPCR / optimization

References

[[1]] Cross NC.Genetic and epigenetic complexity in myeloproliferative neoplasms. Hematology (Am Soc Hematol Educ Program) 2011; 2011: 208-214

[[2]] Vannucchi AM, Antonioli E, Guglielmelli P, Pardanani A, Tefferi A.Clinical correlates of JAK2V617F presence or allele burden in myeloproliferative neoplasms: a critical reappraisal. Leukemia 2008; 22(07) 1299-1307

[[3]] Koren-Michowitz M, Landman J, Cohen Y.et al.JAK2V617F allele burden is associated with transformation to myelofibrosis. Leuk Lymphoma 2012; 53(11) 2210-2213

[[4]] Vannucchi AM, Antonioli E, Guglielmelli P.et al.Clinical profile of homozygous JAK2 617V>F mutation in patients with polycythemia vera or essential thrombocythemia. Blood 2007; 110(03) 840-846

[[5]] Moliterno AR, Ginzburg YZ, Hoffman R.Clinical insights into the origins of thrombosis in myeloproliferative neoplasms. Blood 2021; 137(09) 1145-1153

[[6]] Tefferi A, Lasho TL, Huang J.et al.Low JAK2V617F allele burden in primary myelofibrosis, compared to either a higher allele burden or unmutated status, is associated with inferior overall and leukemia-free survival. Leukemia 2008; 22(04) 756-761

[[7]] Guglielmelli P, Barosi G, Specchia G.et al.Identification of patients with poorer survival in primary myelofibrosis based on the burden of JAK2V617F mutated allele. Blood 2009; 114(08) 1477-1483

[[8]] Nielsen C, Birgens HS, Nordestgaard BG, Kjaer L, Bojesen SE.The JAK2  V617F somatic mutation, mortality and cancer risk in the general population. Haematologica 2011; 96(03) 450-453

[[9]] Nielsen C, Birgens HS, Nordestgaard BG, Bojesen SE.Diagnostic value of JAK2 V617F somatic mutation for myeloproliferative cancer in 49 488 individuals from the general population. Br J Haematol 2013; 160(01) 70-79

[[10]] Genovese G, Kähler AK, Handsaker RE.et al.Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N Engl J Med 2014; 371(26) 2477-2487

[[11]] Jaiswal S, Fontanillas P, Flannick J.et al.Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med 2014; 371(26) 2488-2498

[[12]] Moliterno AR, Kaizer H, Reeves BN.JAK2 V617F allele burden in polycythemia vera: burden of proof. Blood 2023; 141(16) 1934-1942

[[13]] Arber DA, Orazi A, Hasserjian RP.et al.International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: integrating morphologic, clinical, and genomic data. Blood 2022; 140(11) 1200-1228

[[14]] Merker JD, Jones CD, Oh ST, Schrijver I, Gotlib J, Zehnder JL.Design and evaluation of a real-time PCR assay for quantification of JAK2 V617F and wild-type JAK2 transcript levels in the clinical laboratory. J Mol Diagn 2010; 12(01) 58-64

[[15]] Larsen TS, Pallisgaard N, Møller MB, Hasselbalch HC.The JAK2 V617F allele burden in essential thrombocythemia, polycythemia vera and primary myelofibrosis-impact on disease phenotype. Eur J Haematol 2007; 79(06) 508-515

[[16]] Baxter EJ, Scott LM, Campbell PJ.et al; Cancer Genome Project. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365(9464) 1054-1061

[[17]] White HE, Salmon M, Albano F.et al.Standardization of molecular monitoring of CML: results and recommendations from the European treatment and outcome study. Leukemia 2022; 36(07) 1834-1842

[[18]] Cross NC, White HE, Colomer D.et al.Laboratory recommendations for scoring deep molecular responses following treatment for chronic myeloid leukemia. Leukemia 2015; 29(05) 999-1003

[[19]] Shelton DN, Bhagavatula P, Sepulveda N.et al.Performance characteristics of the first Food and Drug Administration (FDA)-cleared digital droplet PCR (ddPCR) assay for BCR:ABL1 monitoring in chronic myelogenous leukemia. PLoS One 2022; 17(03) e0265278

[[20]] Pinheiro LB, Coleman VA, Hindson CM.et al.Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 2012; 84(02) 1003-1011

[[21]] Bhat S, Herrmann J, Armishaw P, Corbisier P, Emslie KR.Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number. Anal Bioanal Chem 2009; 394(02) 457-467

[[22]] Mather K.The analysis of extinction time data in bioassay. Biometrics 1949; 5(02) 127-143

[[23]] Fazekas-de-St G. Fazekas de St Groth. The evaluation of limiting dilution assays. J Immunol Methods 1982; 49(02) R11-R23

[[24]] Majumdar N, Wessel T, Marks J.Digital PCR modeling for maximal sensitivity, dynamic range and measurement precision. PLoS ONE 2015; 10(03) e0118833

[[25]] La Rocca F, Grieco V, Ruggieri V.et al.Superiority of droplet digital PCR over real-time quantitative PCR for JAK2 V617F allele mutational burden assessment in myeloproliferative neoplasms: a retrospective study. Diagnostics (Basel) 2020; 10(03) 143

[[26]] Cordua S, Kjaer L, Skov V, Pallisgaard N, Hasselbalch HC, Ellervik C.Prevalence and phenotypes of JAK2 V617F and calreticulin mutations in a Danish general population. Blood 2019; 134(05) 469-479

[[27]] Link-Lenczowska D, Pallisgaard N, Cordua S.et al.A comparison of qPCR and ddPCR used for quantification of the JAK2 V617F allele burden in Ph negative MPNs. Ann Hematol 2018; 97(12) 2299-2308

[[28]] Lee E, Lee KJ, Park H.et al.Clinical implications of quantitative JAK2 V617F analysis using droplet digital PCR in myeloproliferative neoplasms. Ann Lab Med 2018; 38(02) 147-154

[[29]] Waterhouse M, Follo M, Pfeifer D.et al.Sensitive and accurate quantification of JAK2 V617F mutation in chronic myeloproliferative neoplasms by droplet digital PCR. Ann Hematol 2016; 95(05) 739-744

[[30]] Vogelstein B, Kinzler KW. Digital PCR. Proc Natl Acad Sci U S A 1999; 96(16) 9236-9241

[[31]] Perricone M, Polverelli N, Martinelli G.et al.The relevance of a low JAK2V617F allele burden in clinical practice: a monocentric study. Oncotarget 2017; 8(23) 37239-37249

[[32]] Beer PA, Jones AV, Bench AJ.et al.Clonal diversity in the myeloproliferative neoplasms: independent origins of genetically distinct clones. Br J Haematol 2009; 144(06) 904-908

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