Myeloproliferative Neoplasms - MPNs | Choose the Right Test

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Karner

Kristin Hunt Karner, MD

Associate Professor of Pathology (Clinical), University of Utah

Medical Director, Hematopathology and Molecular Oncology, ARUP Laboratories

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Luo

Matthew X. Luo, MD

Anatomic and Clinical Pathology Resident, University of Utah School of Medicine and ARUP Laboratories

Associate Medical Director, ARUP Consult

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that cause excess blood cell production in the bone marrow and often in the peripheral blood. MPNs include chronic myeloid leukemia (CML, discussed separately), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia (CEL), and myeloproliferative neoplasms, unclassifiable (MPN-U). MPNs are characterized by clonal genetic changes. For example, CML is defined by the presence of the BCR-ABL1 fusion gene, also referred to as the Philadelphia chromosome; the other MPN types are BCR-ABL1 negative. The majority of patients with PV, ET, and PMF have a mutation in the JAK2, CALR, or MPL gene.

Quick Answers for Clinicians

How do myeloproliferative neoplasms differ from myelodysplastic syndromes/myeloproliferative neoplasms?

A myeloproliferative neoplasm (MPN) in the presence of prominent unilineage or multilineage dysplastic features is more appropriately characterized as a myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN). MDS/MPN disorders include chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), juvenile myelomonocytic leukemia (JMML), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), and unclassifiable MDS/MPN.

What is the role of next generation sequencing in the diagnosis of myeloproliferative neoplasms?

Next generation sequencing (NGS) panels that include JAK2, MPL, and CALR genes may be used in place of individual gene tests in the initial diagnostic workup of myeloproliferative neoplasms (MPNs); NGS may also be especially useful to establish clonality in the diagnosis of triple-negative MPNs, in which JAK2, MPL, and CALR gene mutations are not present. Furthermore, NGS can identify multiple spliceosome genes and can also identify additional prognostically relevant mutations. NGS testing is recommended in select patients to evaluate for higher-risk mutations that are associated with disease progression.

Is further testing recommended after a diagnosis of myeloproliferative neoplasm is made?

Coagulation testing to evaluate for acquired von Willebrand disease (VWD) and/or other coagulopathies is recommended in patients with an elevated platelet count and/or unexplained bleeding, and in those who are undergoing high-risk surgical procedures. If hematopoietic cell transplantation is being considered, human leukocyte antigen (HLA) type testing performed on a blood sample is necessary for donor matching.

Indications for Testing

Often, MPN cases are asymptomatic at the time of diagnosis and are detected incidentally through a routine CBC test with an abnormal result or through a finding of splenomegaly with or without hepatomegaly on physical examination. A complete diagnostic workup for an MPN should be considered in the presence of unexplained thrombotic or hemorrhagic events.

If an MPN diagnosis is suspected, further evaluation via additional laboratory testing, molecular testing, cytogenetic analysis, and bone marrow evaluation is indicated.

Diagnostic Criteria

The diagnosis of MPN should be based on the fifth edition of the 2022 World Health Organization (WHO) diagnostic criteria. Criteria include specific findings from the CBC, blood smear, and bone marrow analysis, correlated with clinical history as well as the presence of certain molecular markers and the exclusion of other disorders.

Laboratory Findings in MPNs
Condition	Laboratory Findings
CML	See the ARUP Consult Chronic Myelogenous Leukemia - CML topic
PV	Blood and bone marrow High number of RBCs (high hemoglobin, RBC mass) Hypercellular bone marrow with trilineage growth Increased numbers of mature megakaryocytes with size variation in the bone marrow Genetic No BCR-ABL1 fusion gene JAK2 variants (JAK2 V617F variant present in a majority of cases, JAK2 exon 12 variant may be present, MPL variants rare)
ET	Blood and bone marrow High number of atypical platelets Often normocellular or slightly hypercellular bone marrow Increased numbers of large, mature megakaryocytes with hyperlobated (“staghorn-like”) nuclei in the bone marrow Fibrosis is rare Genetic No BCR-ABL1 fusion gene JAK2, CALR, or MPL variants (JAK2 V617F variant present in a majority of cases, CALR variants may be present, MPL variants present in a small percentage of cases) Other variants may be present in triple-negative (no JAK2, CALR, or MPL) ET
PMF	Blood and bone marrow Prefibrotic/early stage Hypercellular bone marrow with increased granulocytes and markedly atypical megakaryocytes Absent or minimal bone marrow reticulin fibrosis Overt fibrotic stage Leukoerythroblastic blood smear with dacryocytes Normocellular to hypocellular bone marrow with increased atypical megakaryocytes Marked bone marrow reticulin with or without collagen fibrosis Genetic No BCR-ABL1 fusion gene JAK2, CALR, or MPL variants (JAK2 V617F variant present in a majority of cases, CALR variants may be present, MPL variants present in a small percentage of cases) Other variants may be present in triple-negative (no JAK2, CALR, or MPL) PMF
CNL	Blood and bone marrow Sustained mature neutrophilia in peripheral blood Neutrophil-predominant hypercellularity in bone marrow Genetic No BCR-ABL1 fusion gene CSF3R mutations common
CEL, NOS	Blood and bone marrow Eosinophilia Evidence of clonality or increased myeloblasts Hypercellular bone marrow with increased eosinophils Abnormal bone marrow morphology common Genetic No BCR-ABL1 fusion gene No PDGFRA, PDGFRB, or FGFR1 rearrangements (these variants define a different category of neoplasms)
MPN-U	Blood and bone marrow Features of an MPN that do not meet diagnostic criteria for a specific entity (either not fully developed or obscured by advanced stage or another condition) Genetic No BCR-ABL1 fusion gene No PDGFRA, PDGFRB, or FGFR1 rearrangements JAK2, CALR, or MPL variants Other variants may be present in triple-negative (no JAK2, CALR, or MPL) MPN-U
Myeloid and lymphoid neoplasms with PDGFRA, PDGFRB, or FGFR1 variants	See the ARUP Consult Eosinophil-Related Disorders - Eosinophilia topic
NOS, not otherwise specified; RBCs, red blood cells Sources: NCCN, 2022; WHO, 2022

Laboratory Testing

Diagnosis

Along with medical history, symptom assessment, and physical examination, the following laboratory tests are recommended as part of the initial evaluation in the diagnostic workup of MPNs: CBC, peripheral blood smear, comprehensive metabolic panel (CMP) (including uric acid, lactate dehydrogenase [LDH], and liver function tests [LFTs]), serum erythropoietin (EPO), and serum iron studies.

If the results of these tests suggest the possibility of an MPN, bone marrow evaluation, cytogenetics, and molecular testing are indicated. A diagnosis is made when the results of these studies meet the specific diagnostic criteria for a particular MPN subtype. Distinguishing MPN types and grades is essential; treatments vary and long-term clinical outcomes differ significantly among the subtypes.

Testing for BCR-ABL1 Mutation

Testing peripheral blood by fluorescence in situ hybridization (FISH) for the BCR-ABL1 fusion gene is the recommended initial screening test to exclude a diagnosis of CML. Multiplex reverse transcriptase polymerase chain reaction (RT-PCR) testing can also be used to confirm and monitor the BCR-ABL1 fusion gene, but FISH may be more specific and better able to detect unusual breakpoints and cryptic translocations at initial diagnosis. The absence of BCR-ABL1 essentially excludes CML, and testing for other MPN-associated mutations should be performed. For testing specific to CML, see the ARUP Consult Chronic Myelogenous Leukemia topic.

Bone Marrow Evaluation

Bone marrow evaluation is used to confirm an MPN diagnosis and to assess the blast percentage and any evidence of fibrosis, which help to determine the prognosis. Iron staining of a bone marrow aspirate and reticulin and trichrome staining of a bone marrow biopsy are necessary to accurately identify the MPN subtype. Reticulin and trichrome stains also assess the degree of fibrosis. Bone marrow histology should be assessed to rule out disease progression to myelofibrosis before starting cytoreductive therapy.

Cytogenetics

Cytogenetic analysis is important in distinguishing MPN subtypes. The chromosomal analysis also serves to provide evidence of clonality and clonal evolution over time and to assess the success of treatment. Chromosomal evaluation (eg, karyotyping) is performed with or without FISH and should use bone marrow biopsy tissue when possible.

Although specific chromosomal abnormalities have not been identified for MPN subtypes other than CML, abnormal karyotypes may be present in varying degrees among non-CML MPNs. These abnormalities have prognostic significance and may predict the transformation of an MPN to acute myeloid leukemia (AML).

Additional Molecular Testing

Molecular testing is used to assess clonality and to detect MPN-specific mutations. Molecular testing of blood or bone marrow should be performed first for the JAK2 V617F mutation in all patients because it is the most common mutation and occurs in most patients (>90%) with PV and in 60% of patients with ET or PMF. If the JAK2 V617F mutation is not detected, testing for CALR and MPL mutations should follow in patients with ET or PMF and JAK2 exon 12 mutations in patients with PV. In those without JAK2, MPL, or CALR mutations (triple-negative MPNs), a next generation sequencing (NGS) myeloid panel can be useful to establish clonality. Testing using a multigene NGS panel that includes JAK2, CALR, and MPL may be used instead of single-gene tests.

Patients with molecular evidence of PDGFRA, PDGFRB, FGRF1, JAK2, FLT3, ETV6, and other defined rearrangements are considered to have myeloid/lymphoid neoplasms of a different classification from MPN. See the ARUP Consult Eosinophil-Related Disorders - Eosinophilia topic.

Once a diagnosis of MPN has been established, NGS is also recommended to test for additional mutations that may have prognostic importance (eg, ASXL1, CBL, CSF3R, DNM3TA, EZH2, IDH1, IDH2, LINK/SH2B3, SF3B1, SRSF2, TET2, TP53, and U2AF1).

Prognosis

The overall survival of patients with MPNs is variable depending on the subtype, degree of fibrosis, blast percentage, and molecular/cytogenetic profile. Several scoring systems and prognostic models have been developed for the risk stratification of patients with MPNs. See the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology for Myeloproliferative Neoplasms for recommendations on risk stratification.

Monitoring

Medications used in the treatment of MPNs (such as the Janus kinase [JAK] inhibitors ruxolitinib, fedratinib, and pacritinib) may have side effects. See the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology for Myeloproliferative Neoplasms for recommendations on special monitoring considerations for potential disease associations.