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Using Next-Generation Sequencing (NGS) to Derive Key Insights in MPN Testing

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of disorders that include essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF), often referred to as the “classic MPNs.” MPNs are characterized by uncontrolled clonal proliferation of the hematopoietic stem and myeloid progenitor cells1.

People with MPNs have higher risk of developing significant cardiovascular and cerebrovascular complications and development of second cancers, including acute leukemia, which is reported in 1 in 10 MPN patients.2,3

The incidence rate is about 2.7 per 100,000 persons annually. While the incidence is relatively low, the overall prevalence, or number of people living with the disease is much higher due to relatively low mortality rates.4 According to the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute, an estimated ~20,000 people get an MPN each year and there are ~295,000 people living with an MPN in the United States.5

The traditional approach to MPN testing involves molecular analysis of three canonical driver genes, JAK2, CALR, and MPL. Typically, individual single-gene tests are performed sequentially following a standard cascading reflex algorithm to identify disease-associated mutations.

During a recent webinar with The Pathologist, Bevan Tandon MD, a hemato-patholoigst from Pathline, described how this approach to MPN testing can be cumbersome for laboratories. The single-gene testing approach involves many different techniques, each with their own sensitivity and specificity profiles, which can differ across testing modalities. Furthermore, the turnaround time for multiple sequential tests can be extended to weeks.

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As an alternative to cascading single-gene testing, Dr. Tandon described the benefits of next-generation sequencing (NGS), which allows his lab to simultaneously profile JAK2, CALR, MPL, along with many other genes know to be associated with MPNs. Beyond the benefit of fast turnaround time, NGS can help laboratories detect less-common mutation signatures that could be missed using traditional reflex algorithms.

Over the past decade, NGS has helped identify multiple somatic mutations in MPNs and furthered our understanding of disease pathogenesis and clonal evolution. Ongoing research may help expand the use of genomics in MPN prognostic scoring systems.6

To illustrate the benefits of NGS, Dr. Tandon described a research case study from his laboratory using a recent sample derived from a 68yr old male presenting with leukocytosis and thrombocytosis.

Previous testing was limited to only PCR for JAK2 V617F and Sanger Sequencing for JAK2 exon 12 mutations, both of which were negative. More recent testing using fragment analysis was expanded to include CALR, which revealed a 5bp insertion in this canonical gene, commonly associated with Primary Myelofibrosis.

Further testing using NGS confirmed the CALR insertion, but also revealed a cooperating deletion mutation in ASXL1. This finding allowed the lab to refine the sample classification, which is important because studies have shown that co-occurring CALR and ASXL1 mutations may have an impact on disease prognosis.7 Without the use of NGS, molecular testing may have yielded incomplete results, with the ASXL1 mutation potentially being missed.




In a second research case study, Dr. Tandon described the benefit of higher sensitivity with NGS, as compared to traditional Sanger Sequencing. Using a recent sample derived from a 57-year-old female presenting with moderate thrombocytosis, NGS testing detected a positive MPL, p.W515L hotspot mutation at 8.5% variant allele frequency (VAF).

Concurrent testing using Sanger Sequencing failed to detect this canonical mutation, due to the lower analytic sensitivity associated with this method, which is typically observed at 15-20% VAF, as opposed to ≤5% with NGS. Hence, performing Sanger Sequencing alone would have yielded a false-negative result.




In a final example, Dr. Tandon presented a case study where NGS detected a number of alterations, including rare co-mutations involving a Type-1 like CALR mutation together with a MPL oncogenic activating hotspot mutation spanning the canonical 515 codon.

This case highlights the utility of NGS as a multiplex technology capable of assessing multiple gene targets. Dr. Tandon states that the unusual CALR/MPL co-mutation and the other high-molecular-risk alterations may have been missed if testing was limited to traditional, cascaded single-gene testing methods.




You can view the full webinar from Dr. Tandon, where he covers additional research studies involving myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) samples, in addition to recent work using NGS for measurable residual disease (MRD) studies in myeloid neoplasms.

> View Dr. Tandon's Full Webinar



1. Spivak, J.L. Myeloproliferative Neoplasms. N. Engl. J. Med. 2017, 376, 2168–2181.
2. Marchetti, M. Second cancers in MPN: Survival analysis from an international study. Am. J. Haematol. 2020, 95, 295–301.
3. Hultcrantz, M. Risk and Cause of Death in Patients Diagnosed With Myeloproliferative Neoplasms in Sweden Between 1973 and 2005: A Population-Based Study. J. Clin. Oncol. 2015, 33, 2288–2295.
4. McMullin MF. Aetiology of Myeloproliferative Neoplasms. Cancers 2020, 12, 1810
5. https://www.lls.org/research/myeloproliferative-neoplasms-mpn-research-funded-lls 
6. Skov V. Next Generation Sequencing in MPNs. Lessons from the Past and Prospects for Use as Predictors of Prognosis and Treatment Responses. Cancers 2020, 12, 2194
7. Mesa R. A. (2017). NCCN Debuts New Guidelines for Myeloproliferative Neoplasms. Journal of the National Comprehensive Cancer Network : JNCCN, 15(5S), 720–722.



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