Description:
Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal disorders characterized by overproduction of one or more differentiated myeloid lineages.1 These include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The majority of MPN result from somatic mutations in the three driver genes, JAK2, CALR, and MPL, which represent major diagnostic criteria in combination with hematologic and morphological abnormalities.2

Terms such as male and female are used when necessary to refer to sex assigned at birth. 

Background
MPNs are uncommon overlapping blood diseases characterized by the production of 1 or more blood cell lines and include chronic myeloid leukemia (CML), PV, ET, PMF, systemic mastocytosis, chronic eosinophilic leukemia, and others. A common finding in many MPNs is clonality, and a central pathogenic feature is a mutated version of a tyrosine kinase enzyme, such that it is abnormally constitutively activated. The paradigm for use of this information to revolutionize patient management is CML. A unique chromosomal change (Ph) and an accompanying unique gene rearrangement (BCR-ABL) resulting in a continuously activated tyrosine kinase enzyme were identified. These findings led to the development of targeted tyrosine kinase inhibitor drug therapy (imatinib) that produces long-lasting remissions. 

Diagnosis and monitoring of patients with Ph-negative MPNs have been challenging because many of the laboratory and clinical features of the classic forms of these diseases — PV, ET, and PMF — can be mimicked by other conditions such as reactive or secondary erythrocytosis, thrombocytosis, or myeloid fibrosis. Additionally, these entities can be difficult to distinguish on morphologic bone marrow exam, and diagnosis can be complicated by changing disease patterns: PV and ET can evolve into PMF or undergo leukemic transformation. World Health Organization criteria were published as a benchmark for diagnosis in 2001¹ and updated in 2008.²  These have been challenging to use because they involve complex diagnostic algorithms, rely on morphologic assessment of uncertain consistency, and require tests that are not well standardized or widely available, such as endogenous erythroid colony formation. 

In March and April 2005, 4 separate groups using different modes of discovery and different measurement techniques reported the presence of a novel somatic point mutation in the conserved autoinhibitory pseudokinase domain of the gene encoding JAK2 protein in patients with classic MPNs. The mutation caused a valine-to-phenylalanine substitution at amino acid position 617 (JAK2 V617F). Loss of JAK2 autoinhibition, caused by JAK2 V617F, results in constitutive activation of the kinase and in recruitment and phosphorylation of substrate molecules including signal transducers and activators of transcript (STAT) proteins (so-called JAK-Stat signaling). The result is cell proliferation independent of normal growth factor control. These findings were subsequently confirmed, and additional mutations affecting the JAK2 gene — mutations in exon 12 or in complementary pathways such as thrombopoietin-receptor-pathway mutations in MPL exon 10 — were identified. These mutations were seen with varying but reliable frequency in patients with classic MPNs, and with uncommon and erratic frequency in other MPNs. Additionally, unique cases of JAK2 mutations were reported in a subset of patients with Down syndrome‒associated ALL.

Although these mutations were of importance in better understanding the biology of MPNs, they also were of immediate interest as laboratory tools to aid in diagnosis and management of disease. To that end, at least 4 potential intended uses for mutation testing have been considered, including:

a. Diagnosis of patients with clinical, laboratory, or pathologic findings suggesting classic MPNs (PV, ET, or PMF);
b. Diagnosis or selection of treatment for patients with Down syndrome ALL;
c. Phenotyping of disease subtypes in patients with MPNs to establish disease prognosis;
d. Identification, selection, and monitoring of treatment.

Many diagnostic procedures are available for JAK2 testing and MPL mutation testing. Variable analytic and clinical performance has been reported, suggesting that nucleic acid amplification methodologies are more sensitive than mutation sequence analysis. It appears that there can be considerable interassay and interlaboratory variability in testing results.

Regulatory Status 
More than a dozen commercial laboratories currently offer a wide variety of diagnostic procedures for JAK2 testing and MPL mutation testing. These tests are available as laboratory developed procedures under the U.S. Food and Drug Administration (FDA) enforcement discretion policy for laboratorydeveloped tests (LDTs). Clinical laboratories may develop and validate tests in-house and market them as a laboratory service; laboratory-developed tests (LDTs) must meet the general regulatory standards of the Clinical Laboratory Improvement Act (CLIA), and laboratories that offer LDTs must be licensed by CLIA for high-complexity testing. To date, FDA does not require regulatory review of LDTs. 

Policy
Application of coverage criteria is dependent upon an individual’s benefit coverage at the time of the request.

1. For the diagnosis of individuals presenting with clinical, laboratory, or pathological findings suggesting classic forms of myeloproliferative neoplasms (MPN) (e.g., polycythemia vera [PV], essential thrombocythemia [ET], or primary myelofibrosis [PMF]), JAK2, CALR, or MPL mutation testing is consdered MEDICALLY NECESSARY in any of the following situations:
   1. For individuals suspected to have PV who meet at least one of the following testing criteria:
      1. Hemoglobin greater than 16.5 g/dL in men or greater than 16.0 g/dL in women; or hematocrit greater than 49% in men or greater than 48% in women; or increased red cell mass (more than 25% above mean normal predicted value), and no other known cause of erythrocytosis, when measured on two separate occasions.
      2. A bone marrow (BM) biopsy showing hypercellularity for age with trilineage hyperplasia including prominent erythroid, granulocytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes (differences in size).
   2. For individuals suspected to have ET who meet at least one of the following testing criteria:
      1. Platelet count greater than or equal to 450 × 109/L that has persisted for more than three months.
      2. A BM biopsy showing proliferation mainly of the megakaryocyte lineage with increased numbers of enlarged, mature megakaryocytes with hyperlobulated nuclei. No significant increase or left shift in neutrophil granulopoiesis or erythropoiesis and very rarely minor (grade 1) increase in reticulin fibers.
   3. For individuals suspected to have PMF who meet at least one of the following testing criteria:
      1. The individual has demonstrated leukocytosis of greater than or equal to 11 x 109/L on two separate occasions in the absence of other conditions that can cause leukocytosis.
      2. The individual has an enlarged spleen.
      3. A BM biopsy shows megakaryocytic proliferation and atypia, BM fibrosis Grade <2, increased age-adjusted BM cellularity, and granulocytic proliferation; may show erythropoiesis.
      4. A BM biopsy shows presence of megakaryocytic proliferation and atypia, accompanied by either reticulin and/or collagen fibrosis grades 2 or 3.
2. To exclude a diagnosis of chronic myeloid leukemia (CML) for individuals with a suspected MPN, fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR) testing on a peripheral blood sample to detect BCR::ABL1 transcripts is consdered MEDICALLY NECESSARY.
3. For individuals with a clinical suspicion of prePMF or overt PMF who have already tested negative for mutations in JAK2, CALR, or MPL and who do not meet the WHO criteria for BCR-AB1+ CML, PV, ET, myelodysplastic syndromes, or other myeloid neoplasms, screening for mutations in ASXL1, CBL, DNMT3A, EZH2, IDH1/IDH2, RAS, SRSF2, SFS3B1, TET2, TP53, and U2AF1 (see Note 1) is considered MEDICALLY NECESSARY.
4. For individuals diagnosed with Budd-Chiari Syndrome, JAK2, CALR, or MPL mutation testing is considered MEDICALLY NECESSARY.
5. For individuals with normal blood counts and unexplained splanchnic vein thrombosis, screening for JAK2 V617F is considered MEDICALLY NECESSARY.
6. For individuals suspected to have chronic neutrophilic leukemia, testing for CSF3R mutations is considered MEDICALLY NECESSARY.
7. For individuals with a clinical suspicion of mastocytosis, screening for KIT D816V is considered MEDICALLY NECESSARY.

The following does not meet coverage criteria due to a lack of available published scientific literature confirming that the test(s) is/are required and beneficial for the diagnosis and treatment of a patient’s illness.

8. For all other situations not described above, JAK2 tyrosine kinase, CALR, and MPL mutation testing is considered NOT MEDICALLY NECESSARY.

NOTES:
Note 1: For 5 or more gene tests being run on the same platform, please refer to Reimbursement Policy, CAM 235.

Table of Terminology

Term	Definition
ABL	Abelson murine leukemia viral oncogene
aCML	Atypical chronic myeloid leukemia
AHN	Associated Hematologic Neoplasm
ARMS	Amplification refractory mutation system
ASXL1	additional sex combs like 1, transcriptional regulator
BCR	Breakpoint cluster region
BPGM	Bisphosphoglycerate mutase
BSH	British Society for Haematology
CALR	Calreticulin
CBL	Casitas B-lineage lymphoma proto-oncogene
CELNOS	Chronic eosinophilic leukemia, not otherwise specified
CML	Chronic myeloid leukemia
CMML	Chronic myelomonocytic leukemia
CNL	Chronic neutrophilic leukemia
CSF3R	Colony stimulating factor 3 receptor
ddPCR	Digital droplet polymerase chain reaction
DNM3TA	DNA methyltransferase 3 alpha
EASL	European Association for the Study of the Liver
ELN	European Leukemia Net
ESMO	European Society of Medical Oncology
ET	Essential thrombocythemia
EZH2	Enhancer of zeste 2 polycomb repressive complex 2 subunit
FLT3	Fms related receptor tyrosine kinase 3
FLT3-ITD	FLT3- internal duplications
HBA1	Hemoglobin subunit alpha 1
HBA2	Hemoglobin subunit alpha 2
HBB	Hemoglobin subunit beta
HSC	Hematopoietic stem cell
HTLV	Human T-lymphotropic virus type 1
IDH1	Isocitrate dehydrogenase (NADP(+)) 1
IDH2	Isocitrate dehydrogenase (NADP(+)) 2
IR	Ionizing radiation
JAK2	Janus Kinase 2
JMML	Juvenile myelomonocytic leukemia
LNK	Lymphocyte adapter protein
MF	Myelofibrosis
MONU	MPN unclassifiable
MPL	MPL proto-oncogene, thrombopoietin receptor
MPN	Myeloproliferative neoplasms
NCCN	National Comprehensive Cancer Network
PCR	Polymerase chain reaction
PMF	Primary myelofibrosis
PV	Polycythemia vera
RAS	Rat sarcoma virus gene
SETBP	SET binding protein
SF3B1	Splicing factor 3b subunit 1
SH2B3	SH2B adaptor protein 3
SM-AHN	Systemic Mastocytosis with Associated Hematologic Neoplasm
SRSF2	Serine and arginine-rich splicing factor 2
STAT	Signal transducer and activator of transcription
TET2	Tet methylcytosine dioxygenase 2
TP53	Tumor protein p53
U2AF1	U2 small nuclear RNA auxiliary factor 1
WHO	World Health Organization

Rationale
Myeloproliferative neoplasms, including PV, essential thrombocythemia (ET), and primary myelofibrosis (PMF), arise from somatic mutation in hematopoietic stem cell (HSC) that clonally expand resulting in single or multilineage hyperplasia.3 They are relatively rare, affecting 0.84 (PV), 1.03 (ET), and 0.47 (PMF) per 100,000 people worldwide; however, these may not be reflective of its true incidence due to the high heterogeneity of MPN.4

Myeloproliferative neoplasms share features of bone marrow hypercellularity, increased incidence of thrombosis or hemorrhage, and an increased rate of progression to acute myeloid leukemia. Abnormalities in cytokine signaling pathways are common and usually lead to increased JAK-STAT signaling.1 PV is characterized by erythrocytosis with suppressed endogenous erythropoietin production, bone marrow panmyelosis, and JAK2 mutation leading to constitutive activation. ET is defined by thrombocytosis; bone marrow megakaryocytic proliferation; and presence of JAK2, CALR, or MPL mutation. PMF is characterized by bone marrow megakaryocytic proliferation; reticulin and/or collagen fibrosis; and presence of JAK2, CALR, or MPL mutation.2 Mutations in other genes involved in signal transduction (CBL, LNK/SH2B3), chromatin modification (TET2, EZH2, IDH1/2, ASXL1, DNM3TA), RNA splicing (SF3B1, SRSF2, U2AF1), and tumor suppressor function (TP53) have also been reported and are considered “high-risk.”5

The gene JAK2, which stands for “Janus Kinase 2,” is a gene whose mutation is responsible for a significant amount of MPNs. It is a mutation that causes hypersensitivity of hematopoietic progenitor cells to other cytokines, and this mutation typically appears on red blood cells or bone marrow cells. This mutation is often found on exon 12 or 14, and the exon 14 mutation results in a cytokine-independent activation of several regulatory pathways. JAK2 mutations contribute to at least 95% of PV cases, about 50-65% of ET cases, and 60-65% of PMF cases.6-8 

The gene MPL, which encodes a thrombopoietin receptor, also contributes to MPNs. MPL mutations result in a similar phenotype to JAK2 mutations; both result in cytokine-independent growth of their targets. However, MPL mutations are not nearly as common as JAK2 and CALR mutations, casting doubt on the clinical utility for testing. MPL mutations comprise up to 4% of ET cases and 5% of PMF cases.6-8 

The gene CALR encodes calreticulin (or calregulin), which is a Ca2+ binding protein. The mutation typically involves the creation of the incorrect Ca2+ binding region, thereby not allowing the protein to perform its regular duties such as maintaining calcium homeostasis. This results in a similar phenotype to the JAK2 mutation, which is the cytokine-independent activation of regulatory pathways. CALR mutations contribute to approximately 15-25% of ET cases and 20-25% of PMF cases, and about 70% of ET or PMF patients without a JAK2 or MPL mutation have this mutation.6-8 

The significance of JAK2, MPL, CALR and other mutations in the genesis of the MPNs as well as their roles in determining phenotype are unclear.9 However, integrated genomic analyses suggest that regardless of diagnosis or JAK2 mutational status, MPNs are characterized by upregulation of JAK-STAT target genes, demonstrating the central importance of this pathway in the pathogenesis.10 This may lead to development of novel JAK2 therapeutics.11 Thus, mutation analysis at the time of diagnosis has value for determining prognosis as well as individual risk assessment and guide treatment-making decisions.9,12

Neutrophilia, an increase in peripheral blood neutrophils at least two standard deviations above the mean, can be associated with any of the MPNs. In chronic neutrophilic leukemia (CNL), CSF3R mutations have been discovered in most patients with CNL.13,14 A study released in 2013 reported 16 of 27 patients with CNL or atypical chronic myeloid leukemia (aCML) had activating mutations in CSF3R.15 SETBP1 has also been used as a part of comprehensive mutation profiling in distinguishing aCML and chronic myelomonocytic leukemia (CMML). A 2019 NGS study reports significant differences in the profiles of patients with aCML or CMML when comparing TET2, SETBP1, and CSF3R. The researchers conclude, “differential mRNA expression could be detected between both cohorts in a subset of genes (FLT3, CSF3R, and SETBP1 showed the strongest correlation). However, due to high variances in the mRNA expression, the potential utility for the clinic is limited.”16

Dharmawickreme and Witharana (2023) published a 2023 review of allele burden as a valuable biomarker to incorporate in the diagnostic workflow for diagnosis of MPNs. Allele burden refers to the proportion of cells that have a mutation and reflects the ratio of mutant to wild-type JAK2 alleles, providing key insights into disease phenotype and progression. Additionally, the level of JAK2 allele burden differs significantly across the MPN subtypes. For example, a low allele burden is common in ET, and correlates with milder disease phenotypes. A high allele burden is frequently observed in PV and PMF and is associated with more aggressive disease and higher myeloproliferative activity.17 The presence of a JAK2 mutation and the measurement of allele burden may also help differentiate PV from secondary polycythemia (SE), and ET from reactive thrombocytosis (RT).9

Allele burden serves as a promising indicator for prognosis and outcomes for patients. Measuring allele burden can help differentiate between MPN subtypes early, often before clinical manifestation. For example, higher allele burdens are linked to phenotypic expression such as elevated hemoglobin and white blood cells counts, increased spleen size, and more pronounced symptoms in PV and a risk of evolution to post-PV myelofibrosis, PMF or acute myeloid leukemia (AML). Additionally, high allele burden can stratify the risk of thrombosis, as it is associated with a greater risk of thrombotic complications, which are a major cause of mortality in MPNs. Allele burden can also serve as a predictive factor for disease progression and the likelihood of relapse after stem cell transplantation. Thus, measuring allele burden can help serve as a marker to assess measurable residual disease (MRD) following treatment with interferon or hematopoietic cell transplantation.9 

Mutation analysis can be completed with equivalent sensitivity and specificity through use of either peripheral blood granulocytes or bone marrow.9 Currently, qPCR is the most widely used method for allele burden measurement as it offers high-sensitivity. Droplet digital PCR (ddPCR) is emerging as a potentially more precise quantification method. NGS also detects JAK2 and other potential pathogenic mutations.17

Proprietary Testing
In 2017 the FDA approved ipsogen® JAK2 RGQ PCR Kit (FDA, 2017b) to detect Janus Tyrosine Kinase 2 (JAK2) gene mutation G1849T (V617F) with an allele-specific, quantitative, PCR using an amplification refractory mutation system (ARMS). The device marketing authorization was based on data from a clinical study of 473 suspected patients with MPNs, 276 with suspected PV, 98 with suspected ET, and 99 with suspected PMF. The study compared results from the ipsogen JAK2 RGQ PCR Kit to results obtained with independently validated bi-directional sequencing. The study found that the ipsogen JAK2 RGQ PCR Kit test was in 96.8% agreement with the reference method, 100% in positive agreement, and 95.1% in negative agreement, with 458 samples in agreement out of 473. The concordance with each condition was also high; agreement of 90.8% within the ET samples (89/98), 94.9% agreement within the PMF samples (94/99), and 99.6% within the PV samples (275/276). All three conditions had positive agreements of 100%. The authors went on to note that the 15 samples with disagreeing results had mutation levels under the detection capability of bi-directional sequencing. To validate these 15 samples, an independently validated NGS panel was used to compare results with the kit, and all 15 samples were found to test positive, thereby agreeing with the kit. The authors concluded that the kit was accurate for any mutation levels at or above 1%.18

Other proprietary tests are available for mutational analysis in MPN. IntelliGEN® Myeloid is a NGS assay that analyzes fifty genes for somatic mutations that could be useful in providing diagnostic or prognostic information for patients with MDS, AML, or MPN.19 The LeukoVantage® Myeloid Neoplasm Mutation Panel detects myeloid neoplasm-associated mutations in 48 genes associated with AML, MDS, and MPN. The LeukoVantage AML panel can be used to assess AML subclass and prognosis based on genetic abnormalities in NPM1, CEBPA, and RUNX1.20 NeoGenomics offers tests such as the MPN Reflex Test, a sequential testing panel for qualitative detection of JAK2 V617F, JAK2 Exon 12-14, CALR exon 9, and MPL exon 10.21 Centogene has released a Myeloid Tumor Panel which targets 35 genes that are associated with myeloid malignancies which also include AML, MPN, MDS, CML, CMML, and JMML.22

Analytical Validity
Poluben, et al. (2019) analyzed the characteristics of myeloproliferative neoplasms (MPN) in patients exposed to ionizing radiation (IR) from the 1986 Chernobyl accident. 281 patients (90 exposed to radiation, 181 unexposed) were included. JAK2, MPL, and CALR mutations were identified. IR-exposed patients had several different genetic features compared to the unexposed cohort: lower rate of JAK2 V617F mutations (58.4% vs 75.4%), higher rate of type 1-like CALR mutations (12.2% vs 3.1%), higher rate of triple-negative cases (27.8% vs 16.2%), and higher rate of “potentially pathogenic” sequence variants (4.8 vs 3.1). The authors suggested IR-exposed patients as a cohort with “distinct” genomic characteristics.23

Rosenthal, et al. (2021) studied the analytical validity of a 48-gene NGS panel for detecting mutations in myeloid neoplasms. The panel detects detect single nucleotide variations (SNVs), insertions/deletions, and FLT3 internal tandem duplications (FLT3-ITD). 184 samples were analyzed using the 48-gene panel and compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 samples. Analytical validation yielded 99.6% sensitivity and 100% specificity. Concordance of variants detected by the two tested panels was 100%. “Among patients with suspected myeloid neoplasms, 54.5% patients had at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN.” The authors conclude that "the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms even in technically challenging genes."24

Clinical Utility and Validity
An Argentinean study focusing on establishing the frequency of JAK2, MPL, and CALR mutations and comparing their clinical and hematological features corroborates this importance. Mutations of JAK2V617F, JAK2 exon 12, MPL W515L/K and CALR were analyzed in 439 patients with BCR-ABL1-negative MPN, and it was demonstrated that these mutations were present in 94.9% of the cases of PV, 85.5% in patients with essential thrombocythemia (ET), and 85.2% with primary myelofibrosis, leading the researchers to conclude that “the combined genetic tests of these driver mutations are essential for accurate diagnoses of BCR-ABL1-negative MPN.”25

International Consensus Classification for Myeloproliferative Neoplasms
In 2022, a new International Consensus Classification (ICC) was introduced for myeloid neoplasms and acute leukemias by experts involved in prior editions of the WHO classification. The group attempted to refine the diagnostic criteria to show a distinction between the subtypes. They proposed the following criteria for the diagnosis of PV, ET and PMF with subtypes of each.26 

Criteria for PV
Diagnosis of PV requires meeting either all three major criteria, or the first two major criteria and the minor criterion:
Major Criteria

1. Hemoglobin >16.5 g/dL in men; Hemoglobin >16.0 g/dL in women, or Hematocrit >49% in men; Hematocrit >48% in women, or Increased red cell mass (More than 25% above mean normal predicted value)
2. Bone marrow biopsy showing hypercellularity for age with trilineage growth (panmyelosis) including prominent erythroid, granulocytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes (differences in size)
3. Presence of JAK2 V617F or JAK2 exon 12 mutation

Minor Criteria

• Subnormal serum erythropoietin level

Criteria for Post-PV myelofibrosis (MF)

• Diagnosis of Post-PV MF requires meeting all required criteria and at least two additional criteria:

Major Criteria

1. A prior diagnosis of PV
2. Bone marrow fibrosis Grade 2/3

Additional Criteria

1. Anemia or a reduced need for either phlebotomy or cytoreductive treatment to manage erythrocytosis.
2. Presence of leukoerthyroblastosis (immature white and red blood cells in the bloodstream)
3. An increase in palpable splenomegaly of more than 5 cm from baseline or development of a newly palpable spleen.
4. Development of at least 2 (or all 3) of the following constitutional symptoms: weight loss greater than 10% in 6 months, night sweats, unexplainable fever greater than 37.5 degrees Celsius (99.5 degrees Fahrenheit)

Criteria for ET

• Diagnosis of ET requires meeting all four major criteria or the first three major criteria and the minor criterion:

Major Criteria

1. Platelet count ≥450 × 109/L
2. Bone marrow biopsy showing proliferation mainly of the megakaryocyte lineage with increased numbers of enlarged, mature megakaryocytes with hyperlobulated nuclei. No significant increase or left shift in neutrophil granulopoiesis or erythropoiesis.
3. Not meeting WHO criteria for BCR-ABL1+ CML, PV, PMF, myelodysplastic syndromes, or other myeloid neoplasms.
4. Presence of JAK2, CALR, or MPL mutation

Minor Criteria

• Presence of a clonal marker or absence of evidence for reactive thrombocytosis

Criteria for Post-ET MF

• Diagnosis of Post-ET MF requires meeting all required criteria and at least 2 additional criteria

Required Criteria

1. Previous diagnosis of ET
2. Bone marrow fibrosis grade 2/3

Additional criteria

1. Anemia, with a decrease in hemoglobin (b) concentration of more than 2 g/dL from baseline
2. Presence of leukoerythroblastosis (immature white and red blood cells in the bloodstream)
3. An increase in palpable splenomegaly of more than 5 cm from baseline or the development of a newly palpable spleen
4. Elevated LDH levels
5. Development of at least 2 (or all 3) of the following constitutional symptoms: weight loss greater than 10% in 6 months, night sweats, unexplainable fever greater than 37.5 degrees Celsius (99.5 degrees Fahrenheit)

Criteria for PMF, early/prefibrotic stage

•   Diagnosis of prePMF requires meeting all three major criteria, and at least one minor criterion:

Major Criteria

1. Megakaryocytic proliferation and atypia, BM fibrosis Grade <2, increased age-adjusted BM cellularity, granulocytic proliferation, and may show decreased erythropoiesis
2. Not meeting the WHO criteria for BCR-ABL1+ CML, PV, ET, myelodysplastic syndromes, or other myeloid neoplasms
3. Presence of JAK2, CALR, or MPL mutation or in the absence of these mutations, presence of another clonal marker (e.g., ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2, SF3B1), or absence of minor reactive BM reticulin fibrosis

Minor Criteria (presence of one of the following):

1. Anemia not attributed to a comorbid condition
2. Leukocytosis ≥11 × 109/L
3. Palpable splenomegaly
4. LDH increased to above upper normal limit of institutional reference range

Criteria for overt fibrotic stage PMF

• Diagnosis of overt PMF requires meeting all three major criteria, and at least 1 minor criterion (confirmed in 2 consecutive determinations).

Major Criteria

1. Presence of megakaryocytic proliferation and atypia, accompanied by either reticulin and/or collagen fibrosis grades 2 or 3
2. Not meeting WHO criteria for ET, PV, BCR-ABL1+ CML, myelodysplastic syndromes, or other myeloid neoplasms
3. Presence of JAK2, CALR, or MPL mutation or in the absence of these mutations, presence of another clonal marker (e.g., ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2, SF3B1), or absence of reactive myelofibrosis

Minor Criteria

1. Anemia not attributed to a comorbid condition
2. Leukocytosis ≥11 × 109/L
3. Palpable splenomegaly
4. LDH increased to above upper normal limit of institutional reference range
5. Leukoerythroblastosis

These guidelines also list additional “clinicopathologic entities” for MPNs: “chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia, and MPN, unclassifiable (MPN-U).”26

European LeukemiaNet (ELN)
ELN guidelines also recommend “strict adherence” to these guidelines for the three categories of Philadelphia-negative MPNs, (i.e. ET, PV, and MF).27
However, they also recommend “searching” for complementary clonal markers such as ASXL1, EZH2, IDH1/2, and SRSF2 for patients that tested negative for the three driver mutations and have bone marrow features as well as a clinical phenotype consistent with myelofibrosis.27

National Comprehensive Cancer Network (NCCN)
The NCCN Guidelines Version 2.2024 Myeloproliferative Neoplasms recommends molecular testing for JAK2 V617F mutations as part of an initial workup for all patients. If JAK2 mutation testing is negative, molecular testing for CALR and MPL mutations should be performed for patients with suspected ET and MFA, and molecular testing for JAK2 exon 12 should be done for patients who test negative for JAK2 but are suspected of PV. An NGS panel including JAK2, CALR, and MPL may also be used for the workup of all patients. 

The NCCN lists the 2022 edition of the ICC diagnostic criteria. The NCCN does state that NGS “may be useful to establish clonality in selected circumstances (e.g., triple negative non-mutated JAK2, MPL, and CALR).” The NCCN includes a list of somatic mutations with prognostic significance in individuals with MPN that includes the ASXL1, EZH2, RAS, IDH1/2, SRSF2, TP53, U2AF1, DNMT3A, and CBL.

For individuals suspected of MPN, the NCCN recommends excluding a diagnosis of chronic myeloid leukemia: “Fluorescence in situ hybridization (FISH) or a multiplex reverse transcriptase polymerase chain reaction (RT-PCR), if available, on peripheral blood to detect BCR::ABL1 transcripts and exclude the diagnosis of CML is especially recommended for patients with left-shifted leukocytosis and/or thrombocytosis with basophilia.”5

Currently, the NCCN reports that “at the present time, the utility of JAK2 V617F allele burden reduction as a predictor of treatment efficacy remains unclear. . . Therefore, measurement of the JAK2 V617F allele burden is not currently recommended for use in routine clinical practice to guide treatment decisions.”5

British Society for Haematology (BSH)
The BSH recommends testing for CALR for patients suspected of ET and PMR, as CALR mutations account for most patients without either a JAK2 or MPL mutation. The authors found that as many as one third of ET and PMF patients had a mutation in exon 9 of the CALR gene.28

The BSH also published guidelines on the diagnosis of polycythaemia vera. In it, they divide PV into JAK2-positive and JAK2-negative PV. For JAK2-positive PV, the only two diagnostic criteria are as follows:

• “High haematocrit (>0·52 in men, >0·48 in women) OR raised red cell mass (>25% above predicted)”
• “Mutation in JAK2”

For JAK2-negative PV, the diagnostic criteria are as follows (requiring A1-A4, as well as another “A” criteria or two “B” criteria).

• “A1 Raised red cell mass (>25% above predicted) OR haematocrit ≥0·60 in men, ≥0·56 in women”
• “A2 Absence of mutation in JAK2”
• “A3 No cause of secondary erythrocytosis”
• “A4 Bone marrow histology consistent with polycythaemia vera”
• “A5 Palpable splenomegaly”
• “A6 Presence of an acquired genetic abnormality (excluding BCR‐ABL1) in the haematopoietic cells”
• “B1 Thrombocytosis (platelet count >450 × 109 /l)”
• “B2 Neutrophil leucocytosis (neutrophil count >10 × 109 /l in non‐smokers, ≥12.5 × 109 /l in smokers)”
• “B3 Radiological evidence of splenomegaly”
• “B4 Low serum erythropoietin”

The guidelines also note that investigation of erythrocytosis should be undertaken to properly identify the diagnosis. The BSH remarks that EPO receptor mutations may be a primary cause for erythrocytosis and that EGNL1, VHL, and EPAS1 mutations may be a secondary cause. Other hemoglobinopathies caused by mutations in genes such as HBA1, HBA2, HBB, or BPGM may also be a factor.29

In 2021, the BSH published guidelines on the use of genetic tests to diagnose and manage patients with myeloproliferative neoplasms. The following recommendations were made: 

1. “Molecular screening for JAK2, CALR and MPL variants as appropriate is recommended in patients with persistent erythrocytosis or thrombocytosis (GRADE 1B).
2. Screening for JAK2 V617F is recommended in cases with normal blood counts and unexplained splanchnic vein thrombosis (GRADE 1B) and may be considered in selected patients with unexplained cerebral vein thrombosis (GRADE 2C).
    
3. Screening for CALR variants may be considered in patients with splanchnic vein thrombosis or cerebral vein thrombosis (GRADE 2C).
4. Screening for JAK2, CALR and MPL variants should be considered for patients with arterial or unprovoked venous thrombosis who have a mildly or variably elevated haematocrit or platelet count that persists for 2–3 months (GRADE 2C).
5. BCR–ABL1 should be excluded in cases with persistent thrombocytosis negative for JAK2, CALR and MPL variants or with atypical features (GRADE 1B).
6. Younger patients (e.g., under 60 years) with bone marrow histology typical of ET [or myeloproliferative neoplasm, unclassifiable (MPN-U) or suspected prefibrotic MF] where confirmation of a clonal disorder would be useful in view of the patient’s likely long-term disease course and ideally where a broad panel that covers non-canonical variants in JAK2 and MPL and a range of other driver genes is available.
7. Patients with significant thrombocytosis (e.g., platelet count > 600 × 109/l), no reactive cause and borderline bone marrow histology, where cytoreduction would be indicated if there was convincing evidence of a clonal disorder. Examples would include those with an unexplained thrombotic event, particularly younger patients. For older patients without thrombosis, testing may be considered but results must be interpreted with caution in view of the possibility of incidental CH.
8. A myeloid gene panel and cytogenetic analysis (or equivalent) is recommended for patients with bone marrow histology and clinical features consistent with PMF (+/− suggestive features of MDS or MDS/MPN) who test negative for JAK2/CALR/MPL (GRADE 1B).
9. A myeloid gene panel and cytogenetic analysis (or equivalent) is not recommended for most patients with JAK2/CALR/MPL-negative erythrocytosis or thrombocytosis but may be considered in individual cases (GRADE 2C).
10. Myeloid gene panel testing is recommended for MPN cases who test positive for JAK2/CALR/MPL mutations and have additional cytopenias(s) at diagnosis, unexplained ring sideroblasts or other dysplasia, increased blasts (including blastic transformation), peripheral-blood monocytosis or atypical clinical features (GRADE 1B).
11. Myeloid gene panel testing and conventional karyotyping are recommended for all patients with PMF, post-PV or post-ET MF who are candidates for allogeneic stem cell transplant (GRADE 1B).
12. Myeloid gene panel testing should be considered for other patients if the additional genomic data will guide clinical management (GRADE 2C).
13. High-sensitivity assays of mutant allele burden are recommended following post-allogeneic stem cell transplant to monitor for residual disease (GRADE 1C).
14. Quantitative assays of mutant allele burden are not recommended for most MPN patients but may be considered where demonstration of molecular response would influence clinical management (GRADE 2C).
15. Patients with persistent eosinophilia should be investigated initially for FIP1L1–PDGFRA by FISH and/or nested RT-PCR (GRADE 1B).
16. BM cytogenetics or FISH is recommended to screen for other fusion genes, which must then be confirmed by molecular methods (GRADE 1B).
17. Myeloid gene panel and KIT D816V testing should be considered for patients with persistent unexplained eosinophilia who test negative for fusion genes (GRADE 2B).
18. Testing for CSF3R variants, preferably as part of wider myeloid panel, is recommended for all patients with suspected CNL (Grade 2B).
19. Sensitive testing for KIT D816V is recommended for all patients with a clinical suspicion of mastocytosis (GRADE 1B).
20. If negative for KIT D816V, screening for other KIT mutations should be considered for adults (but is recommended for children) (GRADE 1B).
21. Myeloid panel analysis is recommended for patients with advanced SM who are candidates for allogeneic stem cell transplantation (GRADE 1B).
22. Myeloid panel analysis may be considered for other SM patients if the apparent aggressiveness of the disease might influence options for therapy (GRADE 2B).
23. Myeloid panel and/or BM cytogenetics should be considered to characterise the AHN component of SM-AHN (GRADE 2B).
24. BCR–ABL1 should be excluded in all cases of suspected MDS/MPN, and rearrangements associated with MLN-eo should be excluded in cases with eosinophilia (GRADE 1B).
25. Myeloid gene panel analysis and BM cytogenetics or SNP array is recommended for patients diagnosed with MDS/MPN and for cases with suspected MDS/MPN but with indeterminate morphology (GRADE 1B).”30

European Association for the Study of the Liver (EASL)
For myeloproliferative neoplasms, the EASL recommends testing for JAK2 V617F mutations in splanchnic vein thrombosis patients, as well as patients with normal peripheral blood cell counts. If the JAK2 mutation test is negative, a calreticulin mutation test should be performed, and if both are negative, a bone marrow histology analysis should be performed.31

European Society of Medical Oncology (ESMO) 
The ESMO recommends that anyone with a suspected MPN be tested for the three driver mutations (JAK2, CALR, MPL) and that genotyping should be obtained at diagnosis. However, the ESMO states that it is not recommended to repeat testing in follow-up or assessing response to treatment, except for “allogeneic stem-cell transplantation and possibly interferon treatment.” For these two assessments a detection limit of ≤1% is recommended. The ESMO also notes that conventional sequencing methods (PCR, melting analysis) may be used for detecting mutations.32

References 

1. Grinfeld J, Nangalia J, Green AR. Molecular determinants of pathogenesis and clinical phenotype in myeloproliferative neoplasms. Haematologica. 2017;102(1):7-17. doi:10.3324/haematol.2014.113845
2. Rumi E, Cazzola M. Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms. Blood. Feb 9 2017;129(6):680-692. doi:10.1182/blood-2016-10-695957
3. Vainchenker W, Kralovics R. Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood. Feb 9 2017;129(6):667-679. doi:10.1182/blood-2016-10-695940
4. Titmarsh GJ, Duncombe AS, McMullin MF, et al. How common are myeloproliferative neoplasms? A systematic review and meta-analysis. American journal of hematology. Jun 2014;89(6):581-7. 
5. NCCN. NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines: Myeloproliferative Neoplasms v2.2024. NCCN.  https://www.nccn.org/professionals/physician_gls/pdf/mpn.pdf

Coding Section

Code	Number	Description
CPT	81120	IDH1 (isocitrate dehydrogenase 1 [NADP+], soluble) (e.g., glioma), common variants (e.g., R132H, R132C)
	81121	IDH2 (isocitrate dehydrogenase 2 [NADP+], mitochondrial) (e.g., glioma), common variants (e.g., R140W, R172M)
	81175	ASXL1 (additional sex combs like 1, transcriptional regulator) (e.g., myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelomonocytic leukemia), gene analysis; full gene sequence
	81176	ASXL1 (additional sex combs like 1, transcriptional regulator) (e.g., myelodysplastic syndrome, myeloproliferative neoplasms, chronic myelomonocytic leukemia), gene analysis; targeted sequence analysis (e.g., exon 12)
	81206	BCR/ABL1 (t(9;22)) (e.g., chronic myelogenous leukemia) translocation analysis; major breakpoint, qualitative or quantitative
	81207	BCR/ABL1 (t(9;22)) (e.g., chronic myelogenous leukemia) translocation analysis; minor breakpoint, qualitative or quantitative
	81208	BCR/ABL1 (t(9;22)) (e.g., chronic myelogenous leukemia) translocation analysis; other breakpoint, qualitative or quantitative
	81219	CALR (calreticulin) (e.g., myeloproliferative disorders), gene analysis, common variants in exon 9
	81236	EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) (e.g., myelodysplastic syndrome, myeloproliferative neoplasms) gene analysis, full gene sequence
	81237	EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) (e.g., diffuse large B-cell lymphoma) gene analysis, common variant(s) (e.g., codon 646)
	81270	JAK2 (Janus kinase 2) (e.g., myeloproliferative disorder) gene analysis, p.Val617Phe (V617F) variant
	81273	KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) (e.g., mastocytosis), gene analysis, D816 variant(s)
	81275	KRAS (Kirsten rat sarcoma viral oncogene homolog) (e.g., carcinoma) gene analysis; variants in exon 2 (e.g., codons 12 and 13)
	81276	KRAS (Kirsten rat sarcoma viral oncogene homolog) (e.g., carcinoma) gene analysis; additional variant(s) (e.g., codon 61, codon 146)
	81279	JAK2 (Janus kinase 2) (e.g., myeloproliferative disorder) targeted sequence analysis (e.g., exons 12 and 13)
	81311	NRAS (neuroblastoma RAS viral [v-ras] oncogene homolog) (e.g., colorectal carcinoma), gene analysis, variants in exon 2 (e.g., codons 12 and 13) and exon 3 (e.g., codon 61)
	81338	MPL (MPL proto-oncogene, thrombopoietin receptor) (e.g., myeloproliferative disorder) gene analysis; common variants (e.g., W515A, W515K, W515L, W515R)
	81339	MPL (MPL proto-oncogene, thrombopoietin receptor) (e.g., myeloproliferative disorder) gene analysis; sequence analysis, exon 10
	81347	SF3B1 (splicing factor [3b] subunit B1) (e.g., myelodysplastic syndrome/acute myeloid leukemia) gene analysis, common variants (e.g., A672T, E622D, L833F, R625C, R625L)
	81348	SRSF2 (serine and arginine-rich splicing factor 2) (e.g., myelodysplastic syndrome, acute myeloid leukemia) gene analysis, common variants (e.g., P95H, P95L)
	81351	TP53 (tumor protein 53) (e.g., Li-Fraumeni syndrome) gene analysis; full gene sequence
	81352	TP53 (tumor protein 53) (e.g., Li-Fraumeni syndrome) gene analysis; targeted sequence analysis (e.g., 4 oncology)
	81353	TP53 (tumor protein 53) (e.g., Li-Fraumeni syndrome) gene analysis; known familial variant
	81357	U2AF1 (U2 small nuclear RNA auxiliary factor 1) (e.g., myelodysplastic syndrome, acute myeloid leukemia) gene analysis, common variants (e.g., S34F, S34Y, Q157R, Q157P)
	81403	Molecular pathology procedure, Level 4 (e.g., analysis of single exon by DNA sequence analysis, analysis of >10 amplicons using multiplex PCR in 2 or more independent reactions, mutation scanning or duplication/deletion variants of 2-5 exons)
	81405	Molecular pathology procedure, Level 6 (e.g., analysis of 6-10 exons by DNA sequence analysis, mutation scanning or duplication/deletion variants of 11-25 exons, regionally targeted cytogenomic array analysis)
	81432	Hereditary breast cancer-related disorders (e.g., hereditary breast cancer, hereditary ovarian cancer, hereditary endometrial cancer); genomic sequence analysis panel, must include sequencing of at least 10 genes, always including BRCA1, BRCA2, CDH1, MLH1, MSH2, MSH6, PALB2, PTEN, STK11, and TP53
	81442	Noonan spectrum disorders (e.g., Noonan syndrome, cardio-facio-cutaneous syndrome, Costello syndrome, LEOPARD syndrome, Noonan-like syndrome), genomic sequence analysis panel, must include sequencing of at least 12 genes, including BRAF, CBL, HRAS, KRAS, MAP2K1, MAP2K2, NRAS, PTPN11, RAF1, RIT1, SHOC2, and SOS1
	81450	Targeted genomic sequence analysis panel, hematolymphoid neoplasm or disorder, DNA analysis, and RNA analysis when performed, 5-50 genes (e.g., BRAF, CEBPA, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KRAS, KIT, MLL, NRAS, NPM1, NOTCH1), interrogation for sequence variants, and copy number variants or rearrangements, or isoform expression or mRNA expression levels, if performed
	81455	Targeted genomic sequence analysis panel, solid organ or hematolymphoid neoplasm, DNA analysis, and RNA analysis when performed, 51 or greater genes (e.g., ALK, BRAF, CDKN2A, CEBPA, DNMT3A, EGFR, ERBB2, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MLL, NPM1, NRAS, MET, NOTCH1, PDGFRA, PDGFRB, PGR, PIK3CA, PTEN, RET), interrogation for sequence variants and copy number variants or rearrangements, if performed
	81479	Unlisted molecular pathology procedure
	0017U	Oncology (hematolymphoid neoplasia), JAK2 mutation, DNA, PCR amplification of exons 12-14 and sequence analysis, blood or bone marrow, report of JAK2 mutation not detected or detected Proprietary test: JAK2 Mutation Lab/Manufacturer: University of Iowa, Department of Pathology
	0027U	JAK2 (Janus kinase 2) (e.g., myeloproliferative disorder) gene analysis, targeted sequence analysis exons 12-15 Proprietary test: JAK2 Exons 12 to 15 Sequencing Lab/Manufacturer: Mayo Clinic

Procedure and diagnosis codes on Medical Policy documents are included only as a general reference tool for each policy. They may not be all-inclusive.

This medical policy was developed through consideration of peer-reviewed medical literature generally recognized by the relevant medical community, U.S. FDA approval status, nationally accepted standards of medical practice and accepted standards of medical practice in this community, and other nonaffiliated technology evaluation centers, reference to federal regulations, other plan medical policies, and accredited national guidelines.

"Current Procedural Terminology © American Medical Association. All Rights Reserved" 

History From 2013 Forward     

04/17/2025	Annual review, updating criteria #1. Also updating description, table of terminology, rationale, and references.
05/01/2024	Annual review, policy updated for clarity and consistency, adding Coverage Criteria #2, updating rationale, references and coding.
04/12/2023	Annual review, no change to policy intent, but, policy is being rewritten for clarity and consistency. Also updating description, table of terminology, rationale and references.
04/21/2022	Annual review, adding CC#2, 4,5,6 and 7. Also updating coding, rationale and references. Adding table of terminology.
04/22/2021	Annual review, cascade testing removed for clairity. CALR MPL testing added for clarity. Also updating rationale, references and coding.
12/14/2020	Updating Coding Section with 2021 codes
04/14/2020	Annual review, no change to policy intent.
04/05/2019	Annual review, no change to policy intent. Updating title to reflect scope of testing addressed. Also adding policy statement referring to related policy 204115 if testing 5 or more genes.
05/21/2018	correct typo. Interim review adding medical necessity criteria related to testing for primary myelofibrosis: patient has demonstrated leukocytosis of greater or equal to 11x10 to the 9th/ L on two separate occasions in the absence of other conditions that can cause leukocytosis or enlarged spleen", no other changes made.
05/09/2018	Interim review adding medical necessity criteria related to testing for primary myelofibrosis: patient has demonstrated leukocytosis of greater of equal to 11x10 to the 9th/ L on two separate occasions in the absence of other conditions that can cause leukocytosis or enlarged spleen", no other changes made.
12/05/2017	Interim review. Updating policy verbiage and adding new codes
04/27/2017	Interim review, updating to laboratory status and updating policy verbiage for clarity and adding CALR verbiage. Updating title to indicate CALR is being added to policy.
04/26/2017	Updated category to Laboratory. No other changes.
03/01/2017	Annual review, no change to policy intent.
04/27/2016	Interim review, adding additional criteria for JAK2 and MPL testing.
03/15/2016	Annual review, no change to policy intent. Updating background, description, rationale and references.
01/27/2016	Changing review month to correlate with BCA
02/26/2015	Annual review, no change to policy intent, update background, description, guidelines, rationale and references. Adding coding.
02/24/2014	Annual review. Updated title to JAK2 and MPL Mutation Analysis in Myeloproliferative Neoplasms. Updated rationale and references. No change to policy intent.