Implications of Mutation Profiling in Myeloid Malignancies-PART 2: Myeloproliferative Neoplasms and Other Myeloid Malignancies

May 15, 2018

In this second part of our two-part review, we discuss the use of mutation profiling in the diagnosis, prognosis, and treatment of patients with myeloproliferative neoplasms and other myeloid diseases.

Myeloid malignancies arise from the acquisition of somatic mutations among various genes implicated in essential functioning of hematopoietic stem cells and progenitor cells. In this second part of our two-part review, we discuss the use of mutation profiling in the diagnosis, prognosis, and treatment of patients with myeloproliferative neoplasms and other myeloid diseases. We also discuss the entity known as clonal hematopoiesis of indeterminate potential, awareness of which is a result of the increasing availability and improved quality of mutation profiling.

Myeloproliferative Neoplasms: Diagnostic Implications

Mutation analysis can yield valuable diagnostic information in many patients. As demonstrated in Table 1, mutations are highly recurrent across myeloid malignancies. It is important to emphasize that diagnosis should still rely on established clinicopathologic criteria set forth by the World Health Organization (WHO).[1] As an example, for patients with a suspected myeloproliferative neoplasm (MPN), the presence of mutated CALR and/or absence of mutated JAK2 virtually excludes polycythemia vera as a potential diagnosis, with only rare exceptions.[2,3] Additionally, when determining the subtype of MPN, mutations involving ASXL1 and SRSF2 occur more frequently in primary myelofibrosis than polycythemia vera or essential thrombocythemia.[4] In conjunction with a supportive clinical picture and characteristic bone marrow histomorphology, or the absence of a driver mutation (mutated JAK2, CALR, MPL), these mutations can aid in confirming the diagnosis of primary myelofibrosis. Mutations in CSF3R are frequently found in chronic neutrophilic leukemia,[5,6] and they are now incorporated into the latest WHO criteria for diagnosis.[1] Suggested diagnostic workflows for absolute erythrocytosis and thrombocytosis are presented in Figure 1 and Figure 2.

Myeloproliferative Neoplasms: Prognostic Implications

The classic MPNs include the Philadelphia chromosome (BCR-ABL1)–negative MPNs (polycythemia vera, essential thrombocythemia, primary myelofibrosis) and BCR-ABL1–positive chronic myeloid leukemia (CML). The BCR-ABL1–negative MPNs are genetically characterized by the overlapping presence of mutations in three driver genes-JAK2, CALR, and MPL (which encodes for the thrombopoietin receptor)-in the vast majority of patients (> 90%). Regardless of the driver mutation, resultant hyperactivity of JAK-STAT signaling is observed in these patients and is the central pathogenic theme of BCR-ABL1–negative MPNs. Over 50 different mutations involving CALR have been identified; the most common are type 1, a 52–base pair deletion, and type 2, a 5–base pair insertion.[7] MPL W515L/K is present in MPNs, with a frequency of 4% to 5%, and it can coexist with JAK2 V617F.[8] The presence of these MPN driver mutations is now incorporated into the revised 2016 WHO diagnostic criteria for essential thrombocythemia, polycythemia vera, and primary myelofibrosis.[9]

Knowledge of the driver mutation status and of other key genes in MPN patients provides important prognostic information (Table 2). As mentioned, nearly all patients with polycythemia vera harbor mutations in exon 14 (V617F) and exon 12 of JAK2.[10] In 338 patients with polycythemia vera, a high mutant allele burden (ie, > 50% mutant JAK2) was associated with hemoglobin concentration, leukocyte count, age-adjusted bone marrow cellularity, and spleen size. Importantly, allele burden was significantly associated with progression to myelofibrosis, but not with evolution to acute myeloid leukemia or thrombosis risk.[11] A high allele burden was observed in patients with post–polycythemia vera myelofibrosis, but this in itself did not correlate with worse overall survival (OS).[12]

In essential thrombocythemia, mutation profiling can aid in predicting disease-related complication rates. In an international retrospective study of 891 patients with essential thrombocythemia who were followed up for a median of 6.2 years, the presence of JAK2 V617F was associated with increased risk of developing arterial (not venous) thrombosis (hazard ratio [HR], 2.6; P = .009).[13] In another study of 576 essential thrombocythemia patients, 15.5% harbored a CALR mutation, which conferred a longer thrombosis-free survival when compared with JAK2 V617F– and MPL-mutated patients. There was no association of CALR mutation with OS or transformation to myelofibrosis.[14] More recent data from 502 patients with essential thrombocythemia also demonstrate similar survival in JAK2-, MPL-, and CALR-mutated patients.[15] Another study that applied next-generation sequencing (NGS) to 181 essential thrombocythemia patients demonstrated that the number of mutations present was associated with worse OS (HR, 6.6 for 3 mutations). Additionally, multivariate analysis demonstrated that EZH2 and SF3B1 mutations negatively affected OS.[16] Other inherited constitutional genetic variations may also hold useful prognostic information. For example, the presence of DNA repair gene XPD codon 751 Gln/Gln genotype may predispose to the development of leukemic transformation (odds ratio, 4.9), as well as to other nonmyeloid malignancies.[17] Recently, a retrospective study of 208 patients (106 with polycythemia vera and 102 with essential thrombocythemia) with NGS data available demonstrated a higher probability of leukemic transformation in patients harboring the DNMT3A, SRSF2, and IDH1/2 mutations.[18]

In primary myelofibrosis, low JAK2 V617F allele burden has been associated with worse OS. Among 127 JAK2 V617F–positive patients with primary myelofibrosis, the lowest quartile of allele burden had significantly shorter survival compared with other quartiles; this was also seen in JAK2 wild-type patients. Worse outcome was mainly attributed to systemic infections.[19]

Evaluation of driver mutation status is of benefit in assessing prognosis in patients with primary myelofibrosis. In a study of 428 myelofibrosis patients, the median survival in JAK2-, MPL-, and CALR-mutated patients was 5.9, 9.9, and 15.9 years, respectively. Importantly, the worst prognosis was in patients without mutations in these three genes, the so-called “triple-negative” group, with a median survival of only 2.3 years and a higher risk of leukemic transformation.[20] The type of CALR mutation also appears to have distinct prognostic relevance. In a study of 358 primary myelofibrosis patients, those with a type 1 CALR mutation had significantly longer survival when compared with patients who had either JAK2 or type 2 CALR mutations. However, this survival difference disappeared when the Dynamic International Prognostic Scoring System (DIPSS)-Plus score and other mutations were added into the multivariate analysis.[7]

Other mutations have also been shown to have prognostic relevance in primary myelofibrosis. In a study of 879 patients, ASXL1, SRSF2, and EZH2 predicted shorter survival; however, only mutated ASXL1 retained its prognostic significance even when the DIPSS score was included in the analysis. Additionally, leukemia-free survival was negatively impacted by IDH1/2, SRSF2, and possibly ASXL1.[4] A separate study identified the triple-negative and CALR-nonmutated/ASXL1-mutated genotypes as high-risk molecular signatures associated with the worst median survival, 2.5 and 2.3 years, respectively.[21] Taken together, these data identify a high–molecular risk category based on the presence of ASXL1, EZH2, SRSF2, or IDH1/2. In a study of 797 patients with primary myelofibrosis, the presence of more than one high-risk mutation also predicted worse OS and shorter leukemia-free survival when compared with patients with no prognostically detrimental mutations.[22] Other mutations may also share prognostic importance in MPNs. In a series that included 197 patients with polycythemia vera, essential thrombocythemia, or primary myelofibrosis, the presence of TP53 and TET2 were independently associated with lower OS and shorter leukemia-free survival.[23]

Other Myeloid Malignancies: Prognosis

In chronic myelomonocytic leukemia, nonsense/frameshift mutations involving ASXL1 appear to negatively impact survival and improve prognostic value when added to the Mayo chronic myelomonocytic leukemia prognostic model.[24,25] SETBP1 mutations may affect OS, although results are inconsistent.[26] Mutations in RUNX1, on the other hand, do not affect OS but have been associated with shorter leukemia-free survival.[27]

CSF3R is frequently mutated in patients with chronic neutrophilic leukemia, which, as mentioned previously, is important diagnostically.[5,6] Other pathogenic mutations have been observed in chronic neutrophilic leukemia, including CALR, SETBP1, and JAK2 V617F, with unclear prognostic significance.[28] In a series of 14
CSF3R-mutated patients with chronic neutrophilic leukemia, ASXL1 was present in 57% and associated with shortened survival in multivariate analysis.[29]

In patients with systemic mastocytosis, the presence of mutated TET2 is associated with more aggressive forms of the disease, and in a subset of patients with 2008 WHO-defined systemic mastocytosis with associated clonal hematologic non–mast cell diseases, the presence of mutated ASXL1 negatively impacted OS.[30] Additionally, when NGS was applied to patients with systemic mastocytosis, OS was decreased when mutations other than in KIT D816V were observed, most frequently in TET2, SRSF2, ASXL1, CBL, and RUNX1.[31]

Myeloproliferative Neoplasms: Treatment Implications

Primary myelofibrosis

The only approved therapy, ruxolitinib, a JAK1/JAK2 inhibitor, significantly improves splenomegaly and symptom burden in the majority of treated myelofibrosis patients.[32] This clinical benefit has been shown to be irrespective of JAK2 mutation status or the presence of prognostically detrimental mutations (ASXL1, EZH2, SRSF2, IDH1/2).[33] However, when NGS was applied to samples obtained from 95 patients in a phase I/II trial of ruxolitinib, the number of mutations was inversely correlated with spleen response (patients with ≤ 2 mutations had ninefold higher odds of a spleen response than those with ≥ 3 mutations). Additionally, patients with three or more of these mutations had shorter time to treatment discontinuation and shorter OS.[34] The lack of JAK2 V617F does not preclude benefit from ruxolitinib treatment, although patients with a higher mutation burden (≥ 3 mutations) likely have more aggressive disease with worse outcomes even with ruxolitinib treatment.

There is a paucity of data on how mutation status affects hematopoietic stem cell transplantation outcomes. It has been proposed that patients with high-risk mutations, such as ASXL1 or triple-negative disease, should proceed to hematopoietic stem cell transplantation earlier than patients whose high-risk status has been clinically determined and who lack these molecular abnormalities.[35,36] This has not yet been validated in a prospective study.


The cornerstone of CML treatment involves either a first-generation BCR-ABL1 tyrosine kinase inhibitor (TKI) (imatinib) or one of the more potent second-generation TKIs (dasatinib, nilotinib).[37] Importantly, the T315I mutation occurring in the ABL kinase domain of BCR-ABL1 confers imatinib resistance in CML.[38] Second-generation TKIs are also ineffective in patients harboring this specific mutation, as demonstrated in phase I and II clinical trials for dasatinib,[39,40] nilotinib,[41] and bosutinib.[42] Ponatinib is the only available TKI effective against the T315I mutation. In the phase II PACE trial, 449 patients with chronic, acute, or blast-phase CML or BCR-ABL1–positive acute lymphoblastic leukemia resistant to or intolerant of prior TKI therapy were treated with ponatinib, 45 mg once daily. Of those treated, 128 patients harbored the T315I mutation. Patients in all subgroups experienced cytogenetic and molecular responses; in the 270 patients with chronic-phase CML, 56% had a major cytogenetic response at 12 months, including 70% of those with the T315I mutation.[43] This study led to US Food and Drug Administration (FDA) approval of ponatinib for CML and BCR-ABL1–positive acute lymphoblastic leukemia.[44] However, arterial thrombotic events were a treatment-related adverse event observed with ponatinib in the PACE trial, at rates of 2.2%, 0.7%, and 1.6% for cardiovascular, cerebrovascular, and peripheral vascular disease, respectively.[43] This information, combined with postmarketing monitoring, led to an FDA suspension of sales in October 2013, although the suspension was lifted shortly afterwards, following implementation of new safety measures by the manufacturer.[45] Two trials ( identifiers: NCT02467270 and NCT02627677) are currently investigating a reduced dose of ponatinib to optimize its risk/benefit profile. Despite safety concerns, ponatinib represents an efficacious alternative (or bridge) to hematopoietic stem cell transplantation in patients with CML harboring the T315I mutation.

Chronic neutrophilic leukemia

In chronic neutrophilic leukemia, frameshift or truncating mutations of the cytoplasmic tail of CSF3R (S783fs mutation) produce a downstream increase in tyrosine kinase nonreceptor 2 and an Src family kinase.[5,46] In vitro, this mutation confers sensitivity to dasatinib. On the other hand, the presence of proximal mutations (eg, T618I) leads to increases in JAK signaling activity, which demonstrates in vitro sensitivity to ruxolitinib. This has also shown clinical benefit in a report of a single patient with chronic neutrophilic leukemia carrying this proximal mutation.[5,47]

Systemic mastocytosis

A mutation in KIT, most commonly D816V, is a molecular hallmark of systemic mastocytosis and is present in nearly all patients.[48] Unfortunately, this mutation confers resistance to imatinib treatment. Imatinib is currently the only agent that the FDA has approved for use in the treatment of systemic mastocytosis, specifically for non-D816V aggressive systemic mastocytosis.[49] Other TKIs may also be effective in this setting; dasatinib, for example, has shown preclinical activity and clinical efficacy in a small series of patients with systemic mastocytosis.[50] In a large phase II study of 33 patients with systemic mastocytosis, 85% of whom harbored the KIT D816V mutation, the overall response rate was 33%. However, most of the improvement was symptomatic, with only 2 patients achieving complete response.[51] Midostaurin has shown clinical activity in both wild-type and D816V-mutated systemic mastocytosis. In 2016, an international, multicenter, open-label phase II study examined the safety, efficacy, and patient-reported outcomes of midostaurin in 89 patients with advanced systemic mastocytosis. The patients were categorized as having aggressive systemic mastocytosis, systemic mastocytosis with an associated hematologic neoplasm, or mast cell leukemia. Response rates varied between 50% and 75% among the three groups, and response was seen among patients who were positive and negative for KIT D816V mutation. The median OS was significantly longer in responders than in nonresponders (44.4 months vs 15.4 months). Overall, midostaurin was shown to reverse organ damage, decrease splenomegaly and bone marrow mast cell burden, and improve patient quality of life.[52] Based on these promising results, this drug was approved by the FDA for the treatment of aggressive systemic mastocytosis, systemic mastocytosis with associated hematologic neoplasm, and mast cell leukemia in April 2017.[53]

Clonal Hematopoiesis of Indeterminate Potential

As genomic information is becoming increasingly relevant, there is heightened interest in novel assays to detect a variety of alterations. Recently, a novel comprehensive genomic profiling assay of DNA and RNA became available. It is able to detect a variety of clinically relevant genetic alterations (substitutions, insertions/deletions, copy number alterations, and a wide range of gene fusions) in hematologic malignancies.[54] However, with improvements in the quality and availability of mutation profiling, additional mutations are being discovered whose clinical relevance is unclear. One example of this is clonal hematopoiesis of indeterminate potential (CHIP). This newly coined entity is best defined as the presence of somatic mutations in hematologic malignancy–associated genes in blood or bone marrow cells in the absence of cytopenias or hematologic malignancy, or in patients with cytopenias that do not meet the strict criteria for WHO-defined myelodysplastic syndromes. In a patient with unexplained cytopenias, mutation profiling can provide diagnostic assistance (Figure 3). The mutated allele frequency must be > 2% to qualify for the CHIP diagnosis. Compared with myelodysplastic syndromes, CHIP is associated with longer survival and normal blood counts in most cases.[55] The most commonly mutated genes appear to be TET2 and DNMT3A, and the mutations are highly age-correlated.[56] The rate of progression to acute myeloid leukemia is low, at 0.5% to 1% per year, similar to the rate of progression in patients with monoclonal gammopathy of undetermined significance, although higher than in those without known mutations. Guidelines are currently nonexistent for the management of patients in this category. It is proposed that patients in whom mutations are discovered undergo routine surveillance, as in monoclonal gammopathy of undetermined significance, if hematologic abnormalities are present. For clinicians, the counseling and management of patients with CHIP will be an ongoing challenge, but the potential for early intervention in some patients is promising.

As mutation profiling becomes more widespread, it is essential to consider the financial implications of routine testing. The costs of high-throughput gene sequencing, such as NGS, continue to decline and currently cost approximately $200 to $1,000 per sample.[57] Moreover, payers may vary the level of coverage based on clinical circumstances. Poor reimbursement in many cases is likely based on the misconception that these tests are primarily for research purposes and remain of unproven benefit for direct patient care.[58] However, as noted above, genetic assessments have the potential to guide prognosis, therapy, and hematopoietic stem cell transplantation decision making. This personalized genomic information may enable clinicians to provide more effective and high-value care, surpassing the initial costs of molecular testing. Moving forward, payers may be more likely to increase reimbursement for molecular testing as our understanding of mutation profiling in myeloid malignancies expands.


Mutation profiling for patients with myeloid malignancies is already commercially available and widely included in the evaluation and management of many patients in the community. With genomics-directed precision oncology for our patients with myeloid malignancies still in its infancy, the challenge for the practitioner will be in staying updated on clinically relevant advances.

Financial Disclosure:Dr. Rampal serves as a consultant for Agios, Incyte, and Jazz Pharmaceuticals. The other authors have no significant financial interest in or other relationship with the manufacturer of any product or provider of any service mentioned in this article.


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