Multi-center study shows QIAGEN’s ipsogen JAK2 MutaQuant Kit is robust, efficient and sensitive for quantitative detection of the JAK2 V617F mutation

A network study of multiple laboratories finds the ipsogen JAK2 MutaQuant Kit to be robust, efficient and sensitive for quantitative detection of the JAK2 V617F mutation. This study sets the basis for the standardization of molecular techniques for JAK2 V617F determination.

The JAK2 V617F mutation is an important diagnostic biomarker for Philadelphia (Ph)- negative myeloproliferative neoplasms (MPNs), and is a major criterion for the diagnosis of MPNs according to the World Health Organization (WHO) (1–2). This mutation is an acquired, somatic mutation carried by almost all patients with polycythemia vera (PV) and in more than half of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF) (3). Many techniques have been developed over time to determine the presence of JAK2 V617F. However, these techniques present substantial differences in specificity and sensitivity (4–11). Highly sensitive molecular techniques significantly increase the ability to detect small mutated clones with low allele burden (AB) (i.e. under 1% of mutation loads) (4, 6, 8, 10, 11). Therefore, standardization of molecular techniques is needed to provide reliable and comparable molecular results.

In order to evaluate and study the inter- and intra-laboratory variability in JAK2 V617F quantification, identify the most robust assay for standardizing JAK2 V617F molecular testing, and to support consistent interpretation of individual patient analysis results, a network comprised of 19 centers was established (12). All centers collaborated to blindly test DNA samples with different JAK2 V617F AB using both quantitative and qualitative assays. For their assessment, one quantitative and four qualitative assays were evaluated. QIAGEN’s ipsogen JAK2 MutaQuant Kit was used for the quantitative assay, whereas qualitative assays included ipsogen JAK2 MutaSearch Kit. All laboratories using the quantitative ipsogen JAK2 MutaQuant Kit were able to reliably determine the expected JAK2 V617F AB, including low AB ≤ 0.1%. Amongst the qualitative assays, only the ipsogen JAK2 MutaSearch Kit and the ARMS assay (9) was able to detect the 1% AB.

The study concluded that a qualitative approach is not sensitive enough to detect the JAK2 V617F mutation at low AB. The positivity of samples with an AB lower than 1% was not detected by qualitative assays. However, the ipsogen JAK2 MutaQuant CE-IVD kit, which provides quantitative detection of the JAK2 V617F/G1849T mutation using real-time PCR, resulted in a reliable, efficient and sensitive quantitative detection of all mutation loads. The study shows that the quantitative ipsogen JAK2 MutaQuant Kit performs consistently across different platforms, and is a robust method to obtain comparable results. This study sets the basis for the standardization of molecular techniques for JAK2 V617F determination, which will require the employment of approved operating procedures and the use of certificated standards, such as the recent WHO 1st International Reference Panel for Genomic JAK2 V617F (13) to calibrate JAK2 V617F quantitative assays.

QIAGEN offers multiple solutions for quantitative detection of the JAK2 V617F mutation. The ipsogen JAK2 MutaQuant Kit, used in this study (13) and in the collaborative study to evaluate the proposed WHO 1st International Reference Panel for Genomic JAK2 V617F (14), enables sensitive and reliable detection and quantification of the JAK2 V617F/G1849T mutation for in vitro diagnostic use. The latest ipsogen JAK2 RGQ PCR Kit allows detection and quantification of JAK2 V617F/G1849T, with an automated workflow and improved sensitivity of 0.042% limit of detection. In addition to these two ipsogen JAK2 V617F quantitative assays, QIAGEN’s portfolio of solutions for MPN diagnosis also includes an assay to detect CALR mutations, another WHO diagnostic criterion for MPN (2). The ipsogen CALR RGQ PCR Kit reliably detects CALR exon 9 mutations and identifies the two major mutations, Types 1 and 2, in just one real-time PCR step. QIAGEN’s ipsogen CALR RGQ PCR Kit can be used in a synergistic workflow with the ipsogen JAK2 RGQ PCR Kit for reliable, streamlined detection of both CALR and JAK2 V617F mutations from the same patient sample in less than a day, using a single instrument.

Find out more about the broad ipsogen portfolio of oncohematology biomarker solutions available in Europe.

All products mentioned in this article are for in vitro diagnostic use in Europe.

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References:

  1. 1. Tefferi, A. and Vardiman, J.W. (2008) Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia 22: 14–22. Link
  2. 2. Arber, D.A., et al. (2016) The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 127: 2391–2405. Link
  3. 3. Langabeer, S.E. et al. (2015) Molecular diagnostics of myeloproliferative neoplasms. Eur. J. Haematol. 95, 270–279. Link
  4. 4. Bench, A.J., et al. (2013) British Committee for Standards in Haematology. Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations. Br J Haematol. 160: 25–34. Link
  5. 5. Baxter, E.J., et al. (2001) Cancer Genome Project. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 365:1054–1061. Link
  6. 6. McClure, R., Mai, M. and Lasho, T. (2006) Validation of two clinically useful assays for evaluation of JAK2 V617F mutation in chronic myeloproliferative disorders. Leukemia 20: 168–171. Link
  7. 7. Rapado, I., et al. (2008) Validity test study of JAK2 V617F and allele burden quantification in the diagnosis of myeloproliferative diseases. Ann Hematol. 87: 741–749. Link
  8. 8. Lippert, E., et al. (2009) Concordance of assays designed for the quantification of JAK2V617F: a multicenter study. Haematologica 94: 38–45. Link
  9. 9. Chen, Q., et al (2007) Amplification refractory mutation system, a highly sensitive and simple polymerase chain reaction assay, for the detection of JAK2 V617F mutation in chronic myeloproliferative disorders. J Mol Diagn. 9: 272–276. Link
  10. 10. Denys, B., et al. (2010). A real-time polymerase chain reaction assay for rapid, sensitive, and specific quantification of the JAK2V617F mutation using a locked nucleic acid-modified oligonucleotide. J Mol Diagn. 12: 512–519. Link
  11. 11. Jovanovic, J.V. et al. (2013) Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study. Leukemia 27, 2032. Link
  12. 12. Perricone, M., et al. (2017) Assessment of the interlaboratory variability and robustness of JAK2V617F mutation assays: A study involving a consortium of 19 Italian laboratories. Oncotarget 8: 32608-32617. Link
  13. 13. Sanzone, P., et al (2016) Collaborative study to evaluate the proposed WHO 1st International Reference Panel for Genomic JAK2 V617F. Expert committee on biological standardization, Geneva: 17 to 21 October 2016 WHO/BS/2016.2293 Link
Kathryn Collinet

Kathryn Collinet, PhD, is a Technical and Marketing Writer for Personalized Healthcare and Oncology at QIAGEN. She trained as a molecular biologist at the University of Barcelona and the Institute for Research in Biomedicine, where she studied DNA and protein modifications and their influence on chromatin conformation and gene expression. Since 2011 Kathryn has been working in marketing communications for the scientific information and molecular diagnostics industries. Kathryn has a passion for delivering knowledge and insights about molecular and clinical technologies, and their power to impact research and healthcare.

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