Targeted NGS Streamlines Myeloproliferative Neoplasm Diagnostics

The diagnostic landscape of myeloproliferative neoplasms (MPNs) is evolving toward broader, data-rich molecular characterization. Next-generation sequencing (NGS) offers unmatched breadth – revealing driver, co-mutation, and resistance profiles that inform both prognosis and therapy – and is complemented by high-sensitivity polymerase chain reaction (PCR) to detect low-level variants in oncogenic driver genes.

In a webinar supported by Thermo Fisher Scientific, Dr Kritika Krishnamurthy presented:

• enhancements in genomic profiling of MPNs using an amplicon-based myeloid NGS panel

• the relative merits of sequential PCR and targeted NGS testing, illustrated with case studies from her own practice

• a diagnostic algorithm that integrates both NGS and PCR in cases of low variant allele frequency (VAF) in driver genes (e.g., VAF <2%)

Classification of MPNs

MPNs are divided into two main categories; BCR::ABL1-positive MPNs encompassing chronic myeloid leukemia (CML) and BCR::ABL1-negative MPNs which include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), among other subtypes, as shown in Figure 1.

Approximately 90% of MPNs carry mutations in one of three well-defined driver genes: JAK2, CALR, or MPL (2). “These are the primary molecular events driving disease pathogenesis,” explained Dr Krishnamurthy. “Testing for these mutations forms the cornerstone of MPN diagnostics.”

The promise and pitfalls of sequential PCR-based testing

A stepwise PCR testing algorithm – beginning with BCR::ABL1 fusion and followed by JAK2, CALR, and MPL mutational testing – has long been the mainstay of laboratory practice.

The strategy benefits from the dual advantages of economic viability and operational efficiency. Detection of a BCR::ABL1 fusion indicates a CML diagnosis, with no need for further testing. If a BCR::ABL1 negative MPN is suspected, then a series of JAK2, MPL, and CALR testing is required to establish a diagnosis.

Dr Krishnamurthy also highlighted the speed and accessibility of PCR testing as key strengths. Because each assay can be completed within 8 hours, progression through all the steps – from BCR::ABL1 to MPL – can be achieved in around 5 days.

However, Dr Krishnamurthy noted that this approach has limitations in identifying patients with atypical or compound molecular profiles. Hence, relying solely on PCR-based sequential testing risks missing important co-mutations that could guide prognosis or therapy.

Expanding the molecular lens: NCCN guidance on NGS in MPNs

The National Comprehensive Cancer Network (NCCN) guidelines now recommend the use of NGS panels in the diagnostic and prognostic evaluation of MPNs – especially in triple-negative cases (3). NGS helps with confirming clonality, which is essential for establishing the neoplastic nature of the proliferation, distinguishing it from reactive processes, and uncovering co-mutations with prognostic or therapeutic significance.

“These co-mutations carry prognostic weight even in patients who already have identified driver mutations,” said Dr Krishnamurthy.

She concluded that incorporating NGS into MPN workups represents a paradigm shift – from a narrow focus on a few canonical drivers to a comprehensive genomic assessment that better captures disease complexity and supports precision-based management.

Advancing MPN genomic profiling with an amplicon-based myeloid NGS panel

Dr Krishnamurthy presented a series of MPN cases diagnosed on the basis of a targeted amplicon-based NGS panel. In each case, if the patient had been tested according to the sequential PCR algorithm, it would have yielded a different diagnosis.

Case 1: Dual drivers in essential thrombocythemia

A 64-year-old male with persistent thrombocytosis over two years, had a clinical presentation suggestive of essential thrombocythemia. Conventional sequential PCR testing detected a JAK2 V617F mutation, which would typically conclude the diagnostic process under standard testing algorithms.

However, when the same sample was analyzed using a broad NGS myeloid panel, the JAK2 V617F mutation was confirmed at a very low VAF (0.9%). The NGS test also identified an MPL W515L variant at a higher allele fraction (2.3%).

This finding changed the interpretation of the disease biology. Rather than representing a JAK2-driven essential thrombocythemia, the higher-burden MPL mutation was likely the dominant driver, shaping both the patient’s phenotype and clinical trajectory.

Case 2: Uncovering a high-risk co-mutation

A 65-year-old male patient presented with leukocytosis and thrombocytosis, a typical clinical picture of an MPN. Sequential PCR testing identified a JAK2 V617F mutation, while CALR was negative and JAK2 exon 12 and MPL were not assessed. Based on this result alone, the diagnostic process would have concluded with the interpretation of a JAK2-driven neoplasm.

However, when analyzed using NGS, the JAK2 V617F mutation was confirmed at a high VAF (46%), consistent with a significant clonal disease burden. Importantly, NGS also detected a co-occurring IDH2 mutation at a lower allele frequency – an alteration with prognostic and therapeutic implications.

This IDH2 mutation placed the patient in a high molecular risk category, associated with shorter overall survival and a greater likelihood of disease progression compared with cases harboring only JAK2 mutations. Beyond prognosis, this finding carried potential therapeutic relevance: should the disease evolve or fail to respond to standard treatment, IDH inhibitors could represent an alternative treatment strategy.

Case 3: Splicing gene mutation alters prognosis in essential thrombocythemia

An 85-year-old male patient presented with persistent thrombocytosis, consistent with essential thrombocythemia. Standard PCR testing detected a JAK2 V617F mutation, which would typically confirm a JAK2-driven essential thrombocythemia. However, alongside the JAK2 mutation, NGS identified an SF3B1 mutation – a splicing gene alteration associated with inferior overall survival and a higher likelihood of progression to myelofibrosis.

This co-mutation placed the patient in a higher molecular risk category, refining both the prognosis and potential disease trajectory.

Case 4: Establishing clonality in a triple-negative erythrocytosis

PCR-based testing for a 57-year-old female patient presenting with primary erythrocytosis was negative for JAK2, and CALR mutations. The absence of canonical driver mutations initially raised the possibility of a secondary or reactive cause of erythrocytosis. However, clinical evaluation provided no alternative explanation, prompting further molecular assessment with an NGS myeloid panel.

NGS identified a pathogenic DNMT3A variant, confirming clonality and establishing the neoplastic nature of the hematologic proliferation. This finding reclassified the patient’s condition as a triple-negative MPN – likely representing an early or evolving disease phase rather than a reactive process.

Case 5: Low-burden mutations reveal early clonality

In the fifth case, a 63-year-old male patient presented with borderline erythrocytosis and thrombocytosis, yet sequential PCR testing was negative for JAK2, and CALR mutations. To explore the underlying cause, NGS analysis was performed, which identified pathogenic mutations in DNMT3A and TET2 – both at very low VAFs.

These findings confirmed the presence of a clonal hematopoietic process, even though the low allele burden made it unclear whether the mutations represented an incipient MPN or clonal hematopoiesis of indeterminate potential. In either case, the discovery was clinically meaningful: it established molecular clonality where standard PCR testing had found none and provided baseline prognostic information should the disease evolve into a fully manifest MPN.

Case 6: Defining the border between CHIP and early MPN

The final case involved a 55-year-old female patient with erythrocytosis whose PCR testing was negative for JAK2, CALR, and MPL mutations. As in earlier triple-negative cases, further analysis with NGS revealed a pathogenic DNMT3A variant, confirming clonality and establishing that the erythrocytosis was neoplastic rather than reactive in nature.

This finding placed the patient at the borderline between clonal hematopoiesis of indeterminate potential (CHIP) and an early-stage, triple-negative MPN – a distinction that remains diagnostically challenging. While the low-level DNMT3A mutation provided molecular evidence of clonality, it also demonstrated the relationship between CHIP and myeloid neoplasia, emphasizing the importance of longitudinal monitoring and clinical correlation in borderline presentations.

Like the preceding examples, this case demonstrated the value of NGS in uncovering low-level, disease-defining mutations that traditional single-gene assays may overlook, enabling earlier recognition of emerging clonal processes.

NGS followed by PCR for confirmation in cases of low VAF

Dr Krishnamurthy emphasized the importance of confirming ambiguous or low-level variants using other testing methods. Her laboratory has now adopted an integrated workflow, combining the breadth of NGS with the precision of PCR confirmation (Figure 2) (4).

The workflow begins with NGS-based screening for all relevant driver and high-risk mutations. When low-level variants are detected – below standard reporting thresholds – they are confirmed with targeted PCR assays to validate their presence.

This dual approach ensures that no clinically meaningful mutation is overlooked, while maintaining the sensitivity and specificity needed for accurate diagnosis. By reporting the integrated results upfront, her team can simultaneously address diagnosis, clonality, prognosis, and therapeutic guidance – capturing both broad genomic insights from NGS and the high sensitivity of PCR for low-burden variants.

Precision through integration

The integrated NGS–PCR approach that Dr Krishnamurthy advocates ensures comprehensive and reliable results: NGS provides the big picture, capturing disease complexity and informing long-term management, while PCR serves as a confirmatory and fine-tuning tool, validating low-frequency findings critical to early diagnosis and disease monitoring.

In an era where precision and personalization define hematologic oncology, Dr Krishnamurthy’s key message was clear – combining technologies delivers the most accurate, clinically actionable insights, streamlining the path from diagnosis to prognosis to therapy selection for patients with MPNs.

View the webinar

^PMR002050

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