Myeloproliferative Neoplasms - MPN

Primary Authors: Kelley, Todd, MD, MS. Salama, Mohamed E., MD.

Molecular Genetics of BCR-ABL1-Negative Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPNs) are associated with dysregulation of tyrosine kinases, leading to abnormal downstream signaling pathways and increased cellular proliferation. The presence of the BCR-ABL1 mutation characterizes chronic myelogenous leukemia (CML). For BCR-ABL1-negative MPNs, new molecular markers have implications for classification, diagnosis, and prognosis.

Content in tables below based on Chaligné R, et al., 2007; Guglielmelli P, et al., 2009; Klampfl T, et al., 2013; Nangalia, 2013; Schmidt AE, et al, 2012; Vainchenker W, et al., 2011; Vakil E, et al., 2011; Teferri, 2015

JAK2

JAK2 (janus kinase 2)
Mutations	JAK2 V617F mutation Prototypical tyrosine kinase mutation Gain-of-function in exon 14 JAK2 exon 12 mutation Next most common mutation site Missense, insertion, or deletion mutations Observed in JAK2 V617F-negative clones Occurs less frequently in essential thrombocythemia (ET) or primary myelofibrosis (PMF)
Mutation Prevalence	JAK2 V617F mutation Polycythemia vera (PV), 90-95%; ET, ~60%; PMF, ~50% (Teferri, 2015) Mutations rarely found in non-MPNs, such as acute myeloid leukemia (AML), myelodysplastic syndromes (MDS) Mutation is common in the rare MDS/MPN subtype refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) JAK2 exon 12 mutation Present in the majority of patients with PV who are JAK2 V617F-negative, but rarely occurs in PMF, ET
Biology	Cytoplasmic tyrosine kinase activates JAK-STAT pathway, leading to cytokine-independent growth
MPN Disease Association	PV ET PMF
Indications	JAK2 V617F mutation Laboratory tests suggestive of MPN Unexplained erythrocytosis or thrombocytosis; bone marrow fibrosis; BCR-ABL1-negative granulocytosis; unexplained monocytosis or splenomegaly; aquagenic pruritus Symptoms characteristic for MPN Aquagenic pruritus Unusual thrombotic events (eg, splanchnic vein thrombosis, Budd-Chiari syndrome) Unexplained splenomegaly Blood and bone marrow testing are equivalent – performing both is unnecessary JAK2 exon 12 mutation Subnormal erythropoietin and negative JAK2 V617F and CALR testing with symptoms and laboratory test results suggestive of MPN Quantitative testing May be useful in assessment of residual disease after stem cell transplant Enhances diagnostic certainty in cases with low mutant allelic burden
Clinical Implications	JAK2 inhibitors are in development Decrease in mutant JAK2 V617F allelic burden may not correlate with clinical response Utility of monitoring mutant allelic burden is under investigation JAK2 V617F mutation Useful in classifying the disease process as MPN Not useful in classifying specific MPN subtypes High mutant allelic burden – more likely PV Diagnosis based on clinical factors Lower JAK2 V617F allele burden in PMF is associated with poor outcome (Guglielmelli, 2009)
ARUP Tests	JAK2 Gene, V617F Mutation, Qualitative 0051245 JAK2 Gene, V617F Mutation, Quantitative 0040168 JAK2 Exon 12 Mutation Analysis by PCR 2002357 JAK2 Gene, V617F Mutation, Qualitative with Reflex to JAK2 Exon 12 Mutation Analysis by PCR 2012085 JAK2 Gene, V617F Mutation, Qualitative with Reflex to CALR (Calreticulin) Exon 9 Mutation Analysis by PCR with Reflex to MPL codon 515 Mutation Detection by Pyrosequencing, Quantitative 2012084 Myeloid Malignancies Somatic Mutation and Copy Number Analysis Panel 2012182 Myeloid Malignancies Mutation Panel by Next Generation Sequencing 2011117

CALR

CALR (calreticulin)
Mutations	Exon 9 insertions/deletions
Mutation Prevalence	Essential thrombocythemia (ET) – 22% (Teferri, 2015) Primary myelofibrosis (PMF) – 88% of JAK2- and MPL-negative cases (Klampfl, 2013) Mutations occasionally found in refractory anemia with ringed sideroblasts and marked thrombocytosis (RARS-T), chronic myelomonocytic leukemia (CMML), atypical CML
Biology	CALR gene encodes a calcium-binding chaperone protein involved in glycoprotein folding and calcium homeostasis
MPN Disease Association	ET PMF Absent in PV
Indications	JAK2-negative patients when suspicion is high for MPN (excluding PV)
Clinical Implications	Diagnostic for JAK2- and MPL-negative MPN Prognosis – CALR-mutated ET and PMF are associated with increased overall survival when compared with JAK2- and MPL-mutated ET/PMF CALR-mutated ET is associated with a decreased incidence of thrombosis when compared to JAK2-mutated ET Type 2 CALR variant (5-bp TTGTC insertion) – higher platelet counts than type 1 CALR variant (52-bp deletion)
ARUP Available Test	CALR (Calreticulin) Exon 9 Mutation Analysis by PCR 2010673 JAK2 Gene, V617F Mutation, Qualitative with Reflex to CALR (Calreticulin) Exon 9 Mutation Analysis by PCR with Reflex to MPL codon 515 Mutation Detection by Pyrosequencing, Quantitative 2012084 Myeloid Malignancies Somatic Mutation and Copy Number Analysis Panel 2012182 Myeloid Malignancies Mutation Panel by Next Generation Sequencing 2011117

MPL

MPL (myeloproliferative leukemia virus oncogene)
Mutations	MPL W515L or MPL W515K (rarely W515A, W515R) Resides on exon 10 Activates JAK-STAT signaling Observed in JAK2 V617F- and CALR-negative clones
Mutation Prevalence	Essential thrombocythemia (ET) – 3-4% Primary myelofibrosis (PMF) – 8-10% Polycythemia vera (PV) – rare
Biology	Activating mutation of the thrombopoietin receptor causes activation of downstream signaling Contributes to megakaryocytic myeloproliferation
MPN Disease Association	ET PMF
Indications	JAK2 V617F (including exon 12)- and CALR-negative individuals when suspicion for an MPN is high Examples Thrombocytosis and morphologically equivocal for ET Marrow fibrosis and morphologically equivocal for PMF
Clinical Implications	Diagnostic for JAK2-negative MPN
ARUP Tests	MPL codon 515 Mutation Detection by Pyrosequencing, Quantitative 2005545 JAK2 Gene, V617F Mutation, Qualitative with Reflex to CALR (Calreticulin) Exon 9 Mutation Analysis by PCR with Reflex to MPL codon 515 Mutation Detection by Pyrosequencing, Quantitative 2012084 Myeloid Malignancies Somatic Mutation and Copy Number Analysis Panel 2012182 Myeloid Malignancies Mutation Panel by Next Generation Sequencing 2011117

PDGFRA, PDGFRB, and FGFR1

*PDGFRA* (platelet derived growth factor receptor, alpha-polypeptide) *PDGFRB* (platelet derived growth factor receptor, beta-polypeptide) *FGFR1* (fibroblast growth receptor 1)
Mutations	4q12 – PDGFRA 5q31-33 – PDGFRB 8p11 – FGFR1 Translocations cause fusion genes
Mutation Prevalence	FIP1L1-PDGFRA fusion, PDGFRB translocations, or FGFR1 translocations – most common in MPNs associated with eosinophilia FGFR1 gene mutations – also associated with myeloid and lymphoid neoplasms PDGFR gene mutations – also found in chronic myelomonocytic leukemia, atypical chronic myelogenous leukemia, and, rarely, chronic eosinophilic leukemia
Biology	PDGFRA and PDGFRB are receptor kinases that regulate cell growth and proliferation FGFR1 promotes proliferation and survival of eosinophils
MPN Disease Association	Eosinophilic disease
Indications	Unexplained persistent eosinophilia Presence of any of these mutations precludes the diagnosis of eosinophilic leukemia or hypereosinophilic syndrome Cases should be assigned to the corresponding molecularly defined categories Cytogenetics may be negative for FIP1L1-PDGFRA fusion Use fluorescence in situ hybridization (FISH) analysis for detection of mutations if high suspicion FGFR1 mutations usually identified by conventional cytogenetics or FISH
Clinical Implications	Tyrosine kinase inhibitor (TKI) sensitivity (imatinib) FIP1L1-PDGFRA fusion – sensitive PDGFRB translocation – sensitive FGFR1 mutation – not enough data to determine Acquired resistance is common – sorafenib may be alternative inhibitor to use
ARUP Tests	Myeloproliferative Disorders Panel by FISH 2002360 Eosinophilia Panel by FISH 2002378 Chromosome Analysis, Bone Marrow 2002292 PDGFRB FISH for Gleevec Eligibility in Myelodysplastic Syndrome/Myeloproliferative Disease (MDS/MPD) 2012147

KIT

KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog or mast/stem cell growth factor receptor)
Mutations	D816V mutation of the tyrosine kinase domain of the KIT gene Rare (<5%) juxtamembrane KIT mutations
Mutation Prevalence	80% of patients with systemic mastocytosis (KIT D816V mutation)
Biology	KIT (CD117) is a type III tyrosine kinase KIT expression is down-regulated upon differentiation of hematopoietic cells except for mast cells KIT binding to its ligand (stem cell factor) regulates proliferation and maturation
MPN Disease Association	Systemic mastocytosis
Indications	Clinical suspicion for systemic mastocytosis Bone marrow with IHC positive for CD117 and flow cytometry positive for CD117, CD25, and CD2 confirm diagnosis
Clinical Implications	Tyrosine kinase inhibitor (TKI) sensitivity (imatinib) Sensitive KIT D816V-negative Rare KIT juxtamembrane domain mutations Resistant KIT D816V-positive
ARUP Tests	Myeloproliferative Disorders Panel by FISH 2002360 Eosinophilia Panel by FISH 2002378 Chromosome Analysis, Bone Marrow 2002292 CD117 (c-Kit) by Immunohistochemistry 2003806 Leukemia/Lymphoma Phenotyping by Flow Cytometry 2008003 Myeloid Malignancies Somatic Mutation and Copy Number Analysis Panel 2012182 Myeloid Malignancies Mutation Panel by Next Generation Sequencing 2011117

Indications for Testing

Refer to Key Points section

Criteria for Diagnosis

Polycythemia vera (PV)

Must exclude secondary causes of erythrocytosis
2008 WHO diagnostic criteria – must meet both major and one minor criteria or the first major and two minor criteria
- Major criteria
  - Evidence of increased RBC volume, including ≥1 of the following
    - Hgb >18.5 g/dL (men), >16.5 g/dL (women)
    - Hgb or Hct >99th percentile of reference
    - Elevated red cell mass >25% above mean predicted
    - Hgb >17 g/dL (men) or >15 g/dL (women) if associated with sustained increase of ≥2 g/dL not attributed to correction of iron deficiency anemia
  - Presence of JAK2 V617 (90-95%) or JAK2 exon 12 mutations (5-10%)
- Minor criteria
  - Bone marrow trilineage proliferation
  - Subnormal serum erythropoietin level
  - Endogenous erythroid colony growth in vitro

Essential thrombocythemia (ET)

Must exclude other causes of thrombocytosis
- Reactive
  - Anemias
    - Iron deficiency
    - Hemolytic
    - Acute blood loss
- Post splenectomy
- Inflammatory disorders
2008 WHO criteria – must meet all four criteria
- Sustained elevation of platelets ≥450x10⁹/L
  - Defined as at least two measurements, two months apart
- Bone marrow – megakaryocyte proliferation with little or no granulocyte/erythrocyte proliferation
- Does not meet WHO criteria for BCR-ABL1-positive chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, myelodysplastic syndromes, or other myeloid neoplasm
- Demonstration of JAK2 V617F (50%), MPL gene mutations (eg, W515L and W515K), or no evidence of reactive thrombocytosis
Other criteria
- Bone marrow biopsy
  - Normal to hypercellular with increased megakaryocytes
  - Stainable iron
  - Normal red cell blood mass
  - No significant collagen or reticulin fibrosis

Primary myelofibrosis (PMF)

2008 WHO criteria – must meet all three major and two minor criteria
- Major criteria
  - Megakaryocyte proliferation and atypia accompanied by reticulin or collagen fibrosis, or, in the absence of reticulin fibrosis, megakaryocyte changes must be accompanied by increased marrow cellularity
  - Does not meet WHO criteria for BCR-ABL1-positive chronic myelogenous leukemia, polycythemia vera, myelodysplastic syndromes, or other myeloid neoplasm
  - JAK2 V617F or other clonal markers (eg, MPL W515K/L) or no evidence of reactive fibrosis
- Minor criteria
  - Leukoerythroblastosis
  - Increased serum lactate dehydrogenase
  - Anemia
  - Splenomegaly

Laboratory Testing

Initial testing – CBC with differential, erythropoietin (EPO) level, uric acid, lactate dehydrogenase
- Subnormal EPO level highly suggestive of PV if anemia is present
- Normal or increased EPO makes PV unlikely and warrants evaluation for congenital or acquired erythrocytosis
Rule out most common causes of anemia
- Nutritional – vitamin deficiency (B₁₂, folate), iron deficiency
- Gastrointestinal blood loss – fecal blood testing
- Thyroid disease – thyroid-stimulating hormone (TSH)
- Chronic disease – blood urea nitrogen (BUN), creatinine, liver function
Refer to Key Points section

Histology

Noneosinophilic MPN (classic MPN)
- Bone marrow examination with cytogenetic analysis is generally performed after MPN gene testing
  - Not generally necessary if JAK testing is positive and PV suspected
  - May be necessary if ET is suspected since 15% of ET are so called “wild type” (triple negative for current molecular markers)
  - Often necessary for PMF – mutation presence does not distinguish between ET and PMF

Eosinophilic MPN

Molecular genetic testing

Myeloid neoplasms associated with eosinophilia and abnormalities in PDGFRA, PDGFRB, or FGFR1 gene
Cytogenetic and fluorescence in situ hybridization (FISH) analysis for detection of FIP1L1-PDGFFA fusion, PDGFRB (5q33) translocations, or FGFR1 (8p11) translocations

MPN Subtypes, Features, and Diagnostic Criteria

MPN Subtypes, Features, and Diagnostic Criteria
WHO Classification	Features	Laboratory
AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11	Presents as acute myeloid leukemia (AML) Myeloid sarcomas may be present at initial diagnosis or relapse	Morphology – acute myelomonocytic leukemia with increased eosinophils containing immature eosinophilic granules in the bone marrow Peripheral eosinophilia is unusual Diagnosis of AML even if blasts <20% Genetics inv(16)(p13.1q22) or t(16;16)(p13.1;q22) found in most cases inv(16)(p13.1q22) is found in vast majority FISH or PCR may be necessary to document this genetic alteration Secondary cytogenetic abnormalities – +22, +8, del(7q) KIT mutations may be present
Myeloid and lymphoid neoplasms with PDGFRA rearrangement	Most frequently presents as chronic eosinophilic leukemia (CEL), but may present as AML, T-cell lymphoblastic lymphoma (T-LBL), or both Acute transformation can follow CEL presentation Organ infiltration by eosinophils Heart Lungs CNS GI tract Splenomegaly in majority of patients Pronounced male predominance	Morphology Peripheral blood and bone marrow eosinophilia – markedly elevated Typically <20% blasts in peripheral blood and bone marrow Increased bone marrow mast cells common Genetics Absence of BCR-ABL1 fusion gene Most commonly associated with FIP1L1-PDGFRA fusion FISH or PCR is usually necessary to document this genetic alteration; cytogenetic studies are normal Other fusion genes have rarely been identified
Myeloid and lymphoid neoplasms with PDGFRB rearrangement	Presents with features of chronic myelomonocytic leukemia – usually with eosinophilia Splenomegaly in majority of patients Male predominance, but much less marked than PDGFRA-associated neoplasms	Morphology Peripheral leukocytosis Hypercellular bone marrow with typically <20% blasts Increased bone marrow mast cells common Genetics Most common translocation – t(5;12)(q31-33;p13) resulting in formation of ETV6-PDGFRB
Myeloid and lymphoid neoplasms with FGFR1 abnormalities	Often presents with peripheral eosinophilia in the context of lymphadenopathy and lymphoblastic leukemia/lymphoma Slight male predominance	Morphology AML, acute lymphoblastic leukemia (ALL), CEL – usually associated with peripheral blood or bone marrow eosinophilia Genetics Presence of t(8;13)(p11;q12) or a variant translocation at the 8p11 breakpoint leading to FGFR1 rearrangement Secondary cytogenetic abnormalities – trisomy 21 most often observed

In the absence of these molecular markers, chronic eosinophilic leukemia (CEL), not otherwise specified (NOS) (CEL-NOS), or hypereosinophilic syndrome (HES) should be considered
- Diagnosis in both CEL-NOS and HES requires the following
  - Presence of ≥1.5x10⁹/L eosinophil count – peripheral blood
  - Exclusion of secondary eosinophilia
  - Exclusion of other acute or chronic myeloid neoplasm
  - No evidence for phenotypically abnormal or clonal T lymphocytes
- Diagnosis of HES requires the absence of both cytogenetic abnormality and ≥2% peripheral blasts or ≥5% bone marrow blasts

Mast cell MPNs – CD117 (c-Kit), CD25 testing

Prognosis

Classic MPN
- JAK2 V617F – quantitative testing may predict degree of fibrosis, thrombotic tendencies, or survival
- Karyotyping in PMF
  - Unfavorable – complex karyotype or ≥1 abnormality, including +8, -7/7q-, i(17q), -5/5q-, 12p-, inv(3), or 11q23
Eosinophilic MPN
- inv(1) – favorable prognosis, unless associated with KIT mutation
- PDGFRA and PDGFRB mutations – good prognosis with favorable response to tyrosine kinase inhibitors (TKIs)
- FGFR1 mutations – poor prognosis with unclear response to TKIs

Differential Diagnosis

Thrombocytosis
- Primary pulmonary fibrosis and hypertension
- Malignancy
- Infection
- Leukemia
  - Hairy cell leukemia
  - CML
  - Chronic myelomonocytic leukemia
- Connective tissue diseases
- Drugs – corticosteroids, adrenaline
- Hemolysis
- Hyposplenism
- Congenital thrombocytosis (very rare)
Erythrocytosis
- Hypoxia driven processes
  - High altitude habitat
  - Hypoxic lung disease
  - Right to left cardiopulmonary shunts
  - Carbon monoxide poisoning
  - Sleep apnea
  - Renal artery stenosis
- Hypoxia – independent processes
  - Drugs (eg, testosterone)
  - EPO-producing tumors (eg, renal cell, hepatocellular)
  - Renal cysts
  - Renal transplant
Thrombocytopenia
- Myelodysplastic syndrome
- Immune mediated
- Connective tissue disease
- Vasculitis
- Leukemia – AML, ALL
Anemia
- Vitamin/mineral deficiency – B₁₂, iron, folate
- Chronic disease – renal, liver
- Thyroid disease
- Gastrointestinal bleeding – peptic ulcer disease, malignancy
- Infection (eg, parvovirus)
Thromboses
- Hereditary thrombophilias
- Antiphospholipid syndrome
- Malignancy

Myeloproliferative neoplasms (MPN) are a group of slow-growing blood cancers, including chronic myelogenous leukemia (CML). MPNs present with clonal proliferation of abnormal hematopoietic cells that involve bone marrow and peripheral blood.

2008 WHO Classification of Myeloid Neoplasms and Acute Leukemia
Myeloproliferative neoplasms (MPN) CML, BCR-ABL1-positive¹ Chronic neutrophilic leukemia Polycythemia vera¹ Primary myelofibrosis¹ Essential thrombocythemia¹ Chronic eosinophilic leukemia (CEL), not otherwise specified (NOS) Mastocytosis Cutaneous mastocytosis Systemic mastocytosis Mast cell leukemia Mast cell sarcoma Extracutaneous mastocytoma MPN, unclassifiable
Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 gene
Myelodysplastic/myeloproliferative neoplasms Chronic myelomonocytic leukemia Atypical CML, BCR-ABL1-negative Juvenile myelomonocytic leukemia Myelodysplastic/myeloproliferative neoplasm, unclassifiable Refractory anemia with ring sideroblasts associated with marked thrombocytosis²
Myelodysplastic syndromes
Acute myeloid leukemia (AML) and related precursor neoplasms
Acute leukemias of ambiguous lineage
¹Considered classic MPN ²Provisional listing; subject to change

Selected MPNs

Polycythemia vera (PV)

Epidemiology
- Erythroid dominant trilineage proliferation of hematopoietic precursor cells
- Incidence – 0.21-2.27/100,000 (Teferri, 2015)
- Age – peaks in 50s-60s, median age 71
- Sex – M>F (minimal)
Clinical Presentation
- Insidious onset
- Thrombotic complications
  - Deep vein thrombosis
  - Stroke
  - Myocardial infarction
  - Budd-Chiari syndrome
  - Mesenteric ischemia
  - Transient ischemic attacks
- Bleeding complications – often associated with acquired von Willebrand disease
  - Epistaxis
  - Oral mucosal hemorrhage
  - Gastrointestinal hemorrhage
  - Ecchymoses
- Hyperviscosity syndrome – less common
  - Hypertension
  - Headache
  - Dizziness
  - Visual disturbances
  - Claudication
- Erythromelalgia
  - Redness and burning of palms and plantar areas of feet – sometimes progressing to necrosis of digits
- Gouty arthritis
- Pruritus
- Hepatosplenomegaly
- Transformation to myelofibrosis (or spent phase) – short survival
- Transformation to acute leukemia – always fatal without allogeneic bone marrow transplant

Essential thrombocythemia (ET)

Epidemiology
- Thrombocytosis and abnormal megakaryocyte proliferation with clonal proliferation of pluripotent stem cells
- Incidence – 0.01-2.61/100,000 (Teferri, 2015)
- Age – peaks in 50s, median age 71
  - Secondary peak in 20s – mainly females; M<F, 1:2
- Sex – M<F
Clinical Presentation
- Arterial thrombosis
  - Brain
  - Cardiac
  - Extremities
- Venous thrombosis
  - Lower extremity deep vein thrombosis
  - Portal and hepatic vein thrombosis
  - Pulmonary embolism
- Bleeding complications
  - Epistaxis
  - Oral mucosal hemorrhage
  - Gastrointestinal hemorrhage
  - Ecchymosis
  - Acquired von Willebrand disease
- Many patients are asymptomatic and do not require therapy
- Transformation to myelofibrosis (or spent phase) in <10% of cases – short survival
- Transformation to acute leukemia in <5% of cases – always fatal without allogeneic bone marrow transplantation

Primary myelofibrosis (PMF)

Epidemiology
- Clonal stem cell deficit characterized by panmyelosis with intact maturation, progressive bone marrow fibrosis, splenomegaly, multiorgan extramedullary hematopoiesis
- Incidence – 0.3-1.5/100,000 per year
- Age – mean is 67 years
- Sex – M:F, equal
Clinical Presentation
- May be a secondary process in polycythemia vera and essential thrombocythemia
- Shortest survival of all MPNs
- Some with insidious onset and stable; others rapidly progressing
- Acute leukemic transformation common
- Symptoms, physical and laboratory findings
  - Hypercatabolic state – fever, weight loss, night sweats
  - High-output cardiac failure from increased plasma volume
  - Blood abnormality – dacryocytes (tear drops), leukoerythroblastic morphology
  - Other blood abnormalities – anemia, leukocytosis, leukopenia, thrombocytosis, thrombocytopenia, increased circulating blasts
  - Fatigue unrelated to anemia
  - Dyspnea secondary to anemia
  - Petechia secondary to thrombocytopenia
  - Gout
  - Splenomegaly – 100% of patients
  - Hepatomegaly – 50% of patients

Myeloproliferative neoplasms are extremely rare in children.

Polycythemia vera (PV)

Epidemiology

Incidence – rare
Age
- <0.1% with PV are <20 years
- Two peaks – 5-6 years, 10-14 years

Clinical Presentation

Thromboses
- Arterial – stroke
- Venous – Budd-Chiari syndrome
Bleeding disorders
Splenomegaly common
Rubor
Pruritus – especially after hot bath
Erythromelalgia

Diagnosis

Indications for Testing

Refer to Key Points section

Criteria for Diagnosis

See Diagnosis tab for PV criteria

Laboratory Testing

Initial diagnosis – CBC with differential, uric acid, lactate dehydrogenase
Refer to Key Points section

Tests generally appear in the order most useful for common clinical situations. Click on number for test-specific information in the ARUP Laboratory Test Directory.

Erythropoietin 0050227
Method: Quantitative Chemiluminescent Immunoassay

Recommended Use

Initial screening test when evaluating for polycythemia and determining eligibility for erythropoietin therapy in anemia due to chronic renal failure

JAK2 Gene, V617F Mutation, Qualitative 0051245
Method: Polymerase Chain Reaction

Recommended Use

Reasonable screening test for routine evaluation of unexplained erythrocytosis, thrombocytosis, BCR-ABL1-negative granulocytosis, and bone marrow fibrosis

Appropriate screen in individuals with symptoms characteristic for BCR-ABL1-negative MPN

Unusual thrombotic events (eg, splanchnic vein thrombosis, Budd-Chiari)
Aquagenic pruritus
Unexplained splenomegaly

Clinical sensitivity

PV – 95%
ET and PMF – 50%

Limitations

Only one point mutation is detected; exon 12 mutations are not detected

Limit of detection is 0.5% mutant allele

Follow-up

Bone marrow biopsy

Can confirm result with JAK2 V617F mutation, quantitation testing

JAK2 Gene, V617F Mutation, Quantitative 0040168
Method: Polymerase Chain Reaction

Recommended Use

Enhances diagnostic certainty in suspected MPN cases with low mutant allelic burden

Aids in minimal residual disease (MRD) monitoring post stem cell transplant

Clinical sensitivity

PV – 95%
ET and PMF – ~50%

Limitations

Only the one point mutation is detected

Limit of detection is 0.2% mutant allele

Follow-up

Bone marrow biopsy

JAK2 Gene, V617F Mutation, Qualitative with Reflex to JAK2 Exon 12 Mutation Analysis by PCR 2012085
Method: Polymerase Chain Reaction

Recommended Use

Use when diagnosis of PV is suspected

Reflex pattern – if JAK2 V617F is reported as "Not Detected" then JAK2 exon 12 mutation analysis will be added

Limitations

JAK2 V617F qualitative

Only one point mutation is detected
LOD is 0.5% mutant allele

JAK2 exon 12 mutation

Only exon 12 mutations are detected
LOD – 1/1,000 cells

JAK2 Gene, V617F Mutation, Qualitative with Reflex to CALR (Calreticulin) Exon 9 Mutation Analysis by PCR with Reflex to MPL codon 515 Mutation Detection by Pyrosequencing, Quantitative 2012084
Method: Polymerase Chain Reaction/Capillary Electrophoresis/Pyrosequencing

Recommended Use

Use when diagnosis of ET or PMF is suspected

Reflex pattern – if JAK2 V617F is reported as "Not Detected" then CALR exon 9 mutation analysis will be added; if CALR is reported as "Not Detected," then MPL codon 515 mutation detection will be added

Refer to Additional Technical Information document for further content

Limitations

JAK2 V617F qualitative

Only one point mutation is detected
LOD is 0.5% mutant allele

CALR exon 9

Detects only exon 9 indel mutations and does not detect mutations in other regions of the CALR gene
Results must be interpreted in the context of morphological and other relevant data
Test should not be used alone to diagnose malignancy

MPL codon 515

Does not detect mutations in other locations within the MPL gene
LOD – 5% mutant allele

Myeloid Malignancies Somatic Mutation and Copy Number Analysis Panel 2012182
Method: Massively Parallel Sequencing/Genomic Microarray (Oligo-SNP Array)

Recommended Use

Assess for single gene mutations, including substitutions and insertions and deletions that may have diagnostic, prognostic, and/or therapeutic significance in MPN

Combines cytogenomic microarray with an NGS sequencing panel

Panel includes ASXL1, ASXL2, BCOR, BCORL1, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT1, DNMT3A, EED, ELANE, ETNK1, ETV6, EZH2, FAM5C, FLT3, GATA1, GATA2, HNRNPK, IDH1, IDH2, JAK2, JAK3, KDM6A, KIT, KRAS, LUC7L2, MAP2K1, MLL, MPL, NOTCH1, NPM1, NRAS, NSD1, PHF6, PRPF40B, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF1, SF3A1, SF3B1, SMC1A, SMC3, SRSF2, STAG2, SUZ12, TET2, TP53, U2AF1, U2AF2, WT1, ZRSR2

Refer to Additional Technical Information document for further content

Analytical sensitivity – 5% mutant alleles (single nucleotide variants, insertions, and deletions)

Analytical specificity – 100%

Limitations

Mutations may be present below the limit of detection

Not intended to detect minimal residual disease

Myeloid Malignancies Mutation Panel by Next Generation Sequencing 2011117
Method: Massively Parallel Sequencing

Recommended Use

Assess for single gene mutations, including substitutions and insertions and deletions that may have diagnostic, prognostic, and/or therapeutic significance in MPN and MDS/MPN overlap disorders, such as chronic myelomonocytic leukemia

Analytical sensitivity/specificity – 5% mutant alleles (single nucleotide variants, insertions, and deletions); 100%

Refer to Additional Technical Information document for further content

Myeloid panel coordinates table

Limitations

Mutations may not be present below the limit of detection

Not intended to detect MRD

JAK2 Exon 12 Mutation Analysis by PCR 2002357
Method: Polymerase Chain Reaction

Recommended Use

Most appropriate in cases of high suspicion of PV with negative JAK2 V617F and CALR exon 9 mutations

Clinical sensitivity

PV – 2%

Limitations

Only exon 12 mutations are detected

Limit of detection is 1/1,000 cells

CALR (Calreticulin) Exon 9 Mutation Analysis by PCR 2010673
Method: Polymerase Chain Reaction/Capillary Electrophoresis

Recommended Use

Use for diagnostic and prognostic information in patients with MPN when JAK2 testing is negative

Analytical sensitivity – 5% mutant allele burden or 10% heterozygous mutant cells

Analytic specificity – 100%

Limitations

Detects only exon 9 insertion/deletion mutations; does not detect mutations in other regions of the CALR gene

Results must be interpreted in the context of morphological and other relevant data

Test should not be used alone to diagnose malignancy

MPL codon 515 Mutation Detection by Pyrosequencing, Quantitative 2005545
Method: Polymerase Chain Reaction/Quantitative Pyrosequencing

Recommended Use

May be useful when essential thrombocythemia or idiopathic myelofibrosis is suspected in JAK2 V617F-negative individuals

Clinical sensitivity

ET – 3-4%
PMF – 8-10%
PV – very rare

Limitations

Does not detect mutations in other locations within the MPL gene

Limit of detection for this test is 5% mutant allele

Myeloproliferative Disorders Panel by FISH 2002360
Method: Fluorescence in situ Hybridization

Recommended Use

Limited role in the workup of MPN in the setting of otherwise optimal cytogenetic study

Aid in exclusion of cryptic BCR-ABL1 rearrangement in CML and in the exclusion of a PDGFRA abnormality in cases of neoplastic eosinophilia

Probes include BCR-ABL1,PDGFRA, PDGFRB, FGFR1

Refer to Additional Technical Information document for further content

Limitations

Detects only rearrangements targeted by the probes

Does not identify translocation partners of the PDGFRB gene on 5q33 and FGFR1 gene on 8p11

Eosinophilia Panel by FISH 2002378
Method: Fluorescence in situ Hybridization

Recommended Use

Diagnosis, prognosis, and monitoring for newly diagnosed acute or chronic leukemia with eosinophilia

Probes included detect mutations in the following genes – PDGFRA, PDGFRB, FGFR1, and CBFB

Refer to Additional Technical Information document for further content

Limitations

Does not detect rearrangements associated with chronic myelogenous leukemia or identify translocation partners of PDGFRB gene on chromosome 5q33 and FGFR1 gene on chromosome 8p11

Chromosome Analysis, Bone Marrow 2002292
Method: Giemsa Band

Recommended Use

Diagnosis, prognosis, and monitoring of hematopoietic neoplasms

Can also aid in distinguishing this class of hematologic disorders from CML

Follow-up

Repeat testing as clinically indicated to monitor disease progression

Chromosome FISH, Interphase 2002298
Method: Fluorescence in situ Hybridization

Recommended Use

May aid in classification, prognostication, and therapeutic monitoring

Specific FISH probes must be requested and for this indication include probes for PDGFRA, PDGFRB, +8, +9, 20q deletion, monosomy 7/7q deletion, 5q deletion, and 13q deletion

ARUP Oncology FISH Probes menu

Limitations

Limit of detection is probe dependent and around 2-5% in interphase nuclei

Many of these abnormalities can also be detected in myelodysplastic syndromes and AML and are therefore not sufficient for diagnosis but are consistent with the suspected diagnosis (exceptions are mutations in PDGFRA and PDGFRB, which are specific for MPNs)

Follow-up

Repeat testing as clinically indicated to monitor disease progression

PDGFRB FISH for Gleevec Eligibility in Myelodysplastic Syndrome/Myeloproliferative Disease (MDS/MPD) 2012147
Method: Fluorescence in situ Hybridization

Recommended Use

Humanitarian device; authorized by Federal law for use in the qualitative detection of PDGFRB gene rearrangement in patients with myelodysplastic syndrome/myeloproliferative disease (MDS/MPD)

The effectiveness of this device for this use has not been demonstrated; caution: federal law restricts this device to sale by or on the order of a licensed practitioner

PDGFRB FISH for Gleevec Eligibility in MDS/MPD is an in vitro diagnostic test intended for the qualitative detection of PDGFRB gene rearrangement from fresh bone marrow samples of patients with MDS/MPD with a high index of suspicion based on karyotyping showing a 5q31-33 anomaly

The PDGFRB FISH assay is indicated as an aid in the selection of MDS/MPD patients for whom Gleevec (imatinib mesylate) treatment is being considered; this assay is for professional use only and is to be performed at a single laboratory site

CD117 (c-Kit) by Immunohistochemistry 2003806
Method: Immunohistochemistry

Recommended Use

Initial screening test in tumor that is morphologically and clinically suspicious for GIST

Stained and returned to client pathologist; consultation available if needed

CD25 by Immunohistochemistry 2003544
Method: Immunohistochemistry

Recommended Use

Aid in histologic diagnosis of MPN

Stained and returned to client pathologist; consultation available if needed

Leukemia/Lymphoma Phenotyping by Flow Cytometry 2008003
Method: Flow Cytometry

Recommended Use

Aid in evaluation of hematopoietic neoplasms (ie, leukemia, lymphoma)

Specimens include peripheral blood, bone marrow or tissue

Monitor therapy in patients with established diagnosis of hematopoietic neoplasms

Markers selected based on provided clinical history and/or previous test results

Antigens included

T cell: CD1, CD2, CD3, CD4, CD5, CD7, CD8, TCR α-β, TCR γ-δ,, cytoplasmic CD3

B cell: CD10, CD19, CD20, CD22, CD23, CD103, kappa, lambda, FMC7, cytoplasmic kappa, cytoplasmic lambda

Myelo/Mono: CD11b, CD13, CD14 (Mo2), CD14 (MY4), CD15, CD33, CD64, CD117, myeloperoxidase

Misc: CD11c, CD16, CD25, CD30, CD34, CD38, CD41, CD42b, CD45, CD56, CD57, CD61, HLA-DR, glycophorin, TdT, bcl-2, ALK-1, CD123, CD138, CD200, CD26, CD45

Clinical sensitivity – limit of detection 0.01-1.0% depending on phenotype and disease

Oxygen Dissociation (P50) by Hemoximetry (Temporary Referral as of 08/30/16) 2002984
Method: Spectrophotometry/Clark Electrode

Recommended Use

Suspected hemoglobin (Hb) variant with altered oxygen affinity in cyanotic individuals with or without anemia and family history of hemoglobinopathy with polycythemia

Suspected polycythemia vera (PV) when there is isolated polycythemia in individuals who do not exhibit other clinical findings suspicious for PV (eg, leukocytosis, thrombocytosis, or splenomegaly) AND lack a JAK2 gene mutation

Limitations

A simultaneously drawn control specimen from a healthy, nonsmoking individual who is not biologically related to the patient is required for meaningful interpretation

Specimens must be analyzed within 48 hours of collection (P50 value decreases with time)

Will not distinguish between the possible causes for decreased P50 value, which include 2,3-DPG deficiency; high-oxygen-affinity Hb variants; methemoglobinemia; carboxyhemoglobinemia

Decreased P50 values in chronic smokers (carboxyhemoglobinemia) should be interpreted with caution; Hb has higher affinity for carbonmonoxy than for oxygen

P50 values should be correlated with age; HbF may produce a decreased P50 level

von Hippel-Lindau (VHL) Sequencing 2002970
Method: Polymerase Chain Reaction/Sequencing

Recommended Use

Preferred test for evaluation of VHL-associated polycythemia

Use when JAK2 testing is negative and PV is present

Limitations

Large deletions and duplications, deep intronic mutations, and regulatory region mutations are not detected

Rare diagnostic errors may occur due to primer- or probe-site mutations

PV due to causes other than VHL gene mutations are not detected

Guidelines

Gotlib J. World Health Organization-defined eosinophilic disorders: 2014 update on diagnosis, risk stratification, and management. Am J Hematol. 2014; 89(3): 325-37. PubMed

Harrison CN, Bareford D, Butt N, Campbell P, Conneally E, Drummond M, Erber W, Everington T, Green AR, Hall GW, Hunt BJ, Ludlam CA, Murrin R, Nelson-Piercy C, Radia DH, Reilly JT, Van der Walt J, Wilkins B, McMullin MF, British Committee for Standards in Haematology. Guideline for investigation and management of adults and children presenting with a thrombocytosis. Br J Haematol. 2010; 149(3): 352-75. PubMed

Mesa RA, Verstovsek S, Cervantes F, Barosi G, Reilly JT, Dupriez B, Levine R, Le Bousse-Kerdiles M, Wadleigh M, Campbell PJ, Silver RT, Vannucchi AM, Deeg J, Gisslinger H, Thomas D, Odenike O, Solberg LA, Gotlib J, Hexner E, Nimer SD, Kantarjian H, Orazi A, Vardiman JW, Thiele J, Tefferi A, International Working Group for Myelofibrosis Research and Treatment (IWG-MRT). Primary myelofibrosis (PMF), post polycythemia vera myelofibrosis (post-PV MF), post essential thrombocythemia myelofibrosis (post-ET MF), blast phase PMF (PMF-BP): Consensus on terminology by the international working group for myelofibrosis research and Leuk Res. 2007; 31(6): 737-40. PubMed

Protocol for the Examination of Specimens From Patients With Hematopoietic Neoplasms Involving the Bone Marrow. Based on AJCC/UICC TNM, 7th ed. Protocol web posting date: Jun 2012. College of American Pathologists (CAP). Northfield, IL [Accessed: Jun 2015]

Tefferi A, Vardiman JW. Classification and diagnosis of myeloproliferative neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic algorithms. Leukemia. 2008; 22(1): 14-22. PubMed

Vardiman J, Hyjek E. World health organization classification, evaluation, and genetics of the myeloproliferative neoplasm variants. Hematology Am Soc Hematol Educ Program. 2011; 2011: 250-6. PubMed

General References

Cario H, McMullin MF, Pahl HL. Clinical and hematological presentation of children and adolescents with polycythemia vera. Ann Hematol. 2009; 88(8): 713-9. PubMed

Chaligné R, James C, Tonetti C, Besancenot R, Le Couédic JP, Fava F, Mazurier F, Godin I, Maloum K, Larbret F, Lécluse Y, Vainchenker W, Giraudier S. Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis. Blood. 2007; 110(10): 3735-43. PubMed

Gotlib J, Maxson JE, George TI, Tyner JW. The new genetics of chronic neutrophilic leukemia and atypical CML: implications for diagnosis and treatment. Blood. 2013; 122(10): 1707-11. PubMed

Guglielmelli P, Barosi G, Pieri L, Antonioli E, Bosi A, Vannucchi AM. JAK2V617F mutational status and allele burden have little influence on clinical phenotype and prognosis in patients with post-polycythemia vera and post-essential thrombocythemia myelofibrosis. Haematologica. 2009; 94(1): 144-6. PubMed

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Orazi A, Germing U. The myelodysplastic/myeloproliferative neoplasms: myeloproliferative diseases with dysplastic features. Leukemia. 2008; 22(7): 1308-19. PubMed

Patnaik MM, Tefferi A. Chronic Myelomonocytic Leukemia: Focus on Clinical Practice. Mayo Clin Proc. 2016; 91(2): 259-72. PubMed

Rotunno G, Mannarelli C, Guglielmelli P, Pacilli A, Pancrazzi A, Pieri L, Fanelli T, Bosi A, Vannucchi AM, Associazione Italiana per la Ricerca sul Cancro Gruppo Italiano Malattie Mieloproliferative Investigators. Impact of calreticulin mutations on clinical and hematological phenotype and outcome in essential thrombocythemia. Blood. 2014; 123(10): 1552-5. PubMed

Schmidt AE, Oh ST, Education Committee of the Academy of Clinical Laboratory Physicians and Scientists. Pathology consultation on myeloproliferative neoplasms. Am J Clin Pathol. 2012; 138(1): 12-9. PubMed

Tefferi A, Barbui T. Essential Thrombocythemia and Polycythemia Vera: Focus on Clinical Practice. Mayo Clin Proc. 2015; 90(9): 1283-93. PubMed

Tefferi A, Skoda R, Vardiman JW. Myeloproliferative neoplasms: contemporary diagnosis using histology and genetics. Nat Rev Clin Oncol. 2009; 6(11): 627-37. PubMed

Tefferi A, Thiele J, Vannucchi AM, Barbui T. An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms. Leukemia. 2014; 28(7): 1407-13. PubMed

Tefferi A, Vainchenker W. Myeloproliferative neoplasms: molecular pathophysiology, essential clinical understanding, and treatment strategies. J Clin Oncol. 2011; 29(5): 573-82. PubMed

Tefferi A. Polycythemia vera and essential thrombocythemia: 2012 update on diagnosis, risk stratification, and management. Am J Hematol. 2012; 87(3): 285-93. PubMed

Tefferi A. Primary myelofibrosis: 2013 update on diagnosis, risk-stratification, and management. Am J Hematol. 2013; 88(2): 141-50. PubMed

Vainchenker W, Delhommeau F, Constantinescu SN, Bernard OA. New mutations and pathogenesis of myeloproliferative neoplasms. Blood. 2011; 118(7): 1723-35. PubMed

Vakil E, Tefferi A. BCR-ABL1--negative myeloproliferative neoplasms: a review of molecular biology, diagnosis, and treatment. Clin Lymphoma Myeloma Leuk. 2011; 11 Suppl 1: S37-45. PubMed

Vardiman J, et al. Myeloproliferative Neoplasms. In Swerdlow SH, et al. WHO Classification of Tumours of Haematoietic and Lymphoid Tissues, 4th. Lyon, France: IARC Press, 2008.

References from the ARUP Institute for Clinical and Experimental Pathology®

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Bruchova H, Yoon D, Agarwal AM, Mendell J, Prchal JT. Regulated expression of microRNAs in normal and polycythemia vera erythropoiesis. Exp Hematol. 2007; 35(11): 1657-67. PubMed

Bruchova H, Yoon D, Agarwal AM, Swierczek S, Prchal JT. Erythropoiesis in polycythemia vera is hyper-proliferative and has accelerated maturation. Blood Cells Mol Dis. 2009; 43(1): 81-7. PubMed

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Chen GL, Liu E, Naidoo K, Popat U, Coetzer TL, Prchal JT. Idiopathic myelofibrosis without dacryocytes. Haematologica. 2006; 91(6 Suppl): ECR29. PubMed

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