Descriptions:
Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal disorders characterized by overproduction of one or more differentiated myeloid lineages (Grinfeld et al., 2017). These include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The majority of MPN result from somatic mutations in the 3 driver genes, JAK2, CALR, and MPL, which represent major diagnostic criteria in combination with hematologic and morphological abnormalities (Rumi & Cazzola, 2017).

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 considered 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 3 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, without reticulin fibrosis >grade 1, accompanied by increased age-adjusted BM cellularity, granulocytic proliferation, and often decreased 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. 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 clonal markers ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2, and SFS3B1 (see Note 1) is considered MEDICALLY NECESSARY.
3. For individuals diagnosed with Budd-Chiari Syndrome, JAK2, CALR, or MPL mutation testing is considered MEDICALLY NECESSARY.
4. For individuals with normal blood counts and unexplained splanchnic vein thrombosis, screening for JAK2 V617F is considered MEDICALLY NECESSARY.
5. For individuals suspected to have chronic neutrophilic leukemia, testing for CSF3R mutations is considered MEDICALLY NECESSARY.
6. 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.

7. 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.

Rationale
Myeloproliferative neoplasms, including polycythemia vera (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 (Vainchenker & Kralovics, 2017). 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 (Titmarsh et al., 2014). 

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 (Grinfeld et al., 2017). 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 (Rumi & Cazzola, 2017). 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” (NCCN, 2019, 2022)

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 (Tefferi, 2022a, 2023a, 2023b). 

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 (Tefferi, 2022a, 2023a, 2023b). 

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 (Tefferi, 2022a, 2023a, 2023b). 

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 (Tefferi, 2022b). 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 (Rampal et al., 2014). This may lead to development of novel JAK2 therapeutics (Silvennoinen & Hubbard, 2015). Thus, mutation analysis at the time of diagnosis has value for determining prognosis as well as individual risk assessment and guide treatment-making decisions (Hussein et al., 2013; Tefferi, 2022b).

Neutrophilia, an increase in peripheral blood neutrophils at least two standard deviations above the mean, can be associated with any the MPNs. In chronic neutrophilic leukemia (CNL), CSF3R mutations have been discovered in most patients with CNL (Coates, 2022; Tefferi et al., 2014). A study released in 2013 reported 16 of 27 patients with CNL or atypical chronic myeloid leukemia (aCML) had activating mutations in CSF3R (Maxson et al., 2013). 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” (Faisal et al., 2019).

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, polymerase chain reaction (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% (FDA, 2017a).

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 (labcorp, 2023). 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 (Quest_Diagnostics, 2020). NeoGenomics offers two tests which include the MPN Reflex Test and NeoTYPE® Myeloid Disorders Profile. The MPN Reflex Test is a sequential testing panel for qualitative detection of JAK2 V617F, JAK2 Exon 12-14, CALR exon 9, and MPL exon 10 (NeoGenomics, 2022a). NeoTYPE® Myeloid Disorders Profile is a 63 gene panel that targets known mutations associated with AML, MPN, MDS, CML, chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) (NeoGenomics, 2022b). 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 (Centogene, 2022).

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 (Poluben et al., 2019).

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 2 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" (Rosenthal et al., 2021).

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 polycythemia vera (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” (Ojeda et al., 2018).

World Health Organization (WHO) 
The 2017 edition of the World Health Organization’s classification of myeloid neoplasm and acute leukemia proposed the following criteria for the diagnosis of PV, ET and PMF.

WHO Criteria for PV
Diagnosis of PV requires meeting either all 3 major criteria, or the first 2 major criteria and the minor criterion:

Major Criteria

1. Hemoglobin >16.5 g/dL in men; Hemoglobin >16.0 g/dL in women, or diHematocrit >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 JAK2V617F or JAK2 exon 12 mutation

Minor Criteria
Subnormal serum erythropoietin level

WHO Criteria for ET
Diagnosis of ET requires meeting all 4 major criteria or the first 3 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 and very rarely minor (grade 1) increase in reticulin fibers
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

WHO Criteria for PrePMF

Diagnosis of prePMF requires meeting all 3 major criteria, and at least 1 minor criterion:

Major Criteria

1. Megakaryocytic proliferation and atypia, without reticulin fibrosis >grade 1, accompanied by increased age-adjusted BM cellularity, granulocytic proliferation, and often 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

The minor criteria must be confirmed in 2 consecutive determinations.

WHO Criteria for Overt PMF

Diagnosis of overt PMF requires meeting all 3 major criteria, and at least 1 minor criterion

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 (T. Barbui et al., 2018)

These guidelines also list four additional “clinicopathologic entities” for MPNs: “chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), chronic eosinophilic leukemia, not otherwise specified (CELNOS) and MPN, unclassifiable (MPN-U)”. The guidelines note that although CSF3R mutations are “specific” to WHO-defined CNL, they also remark that “the presence of a membrane proximal CSF3R mutation in a patient with neutrophilic granulocytosis should be sufficient for the diagnosis of CNL, regardless of the degree of leukocytosis” (T. Barbui et al., 2018).

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) (Tiziano Barbui et al., 2018).

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 (Tiziano Barbui et al., 2018).

National Comprehensive Cancer Network (NCCN)
The NCCN Guidelines Version 3.2022 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 ET and PMF patients, 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. The NCCN follows the 2017 edition of the WHO diagnostic criteria for all three conditions. 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, IDH1/2, SRSF2, TP53, and U2AF1 Q157.

Finally, the NCCN recommends following the 2017 WHO diagnostic criteria to diagnose MPNs (NCCN, 2022).

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 (Harrison et al., 2014).

The BSH also published guidelines on the diagnosis of polycythaemia vera (PV). 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 (McMullin et al., 2019).

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)” (Cross et al., 2021).

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 (EASL, 2016).

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 (Vannucchi et al., 2015).

Table of Terminology

References 

1. Barbui, T., Tefferi, A., Vannucchi, A. M., Passamonti, F., Silver, R. T., Hoffman, R., Verstovsek, S., Mesa, R., Kiladjian, J.-J., Hehlmann, R., Reiter, A., Cervantes, F., Harrison, C., Mc Mullin, M. F., Hasselbalch, H. C., Koschmieder, S., Marchetti, M., Bacigalupo, A., Finazzi, G., . . . Barosi, G. (2018). Philadelphia chromosome-negative classical myeloproliferative neoplasms: revised management recommendations from European LeukemiaNet. Leukemia, 32(5), 1057-1069. https://doi.org/10.1038/s41375-018-0077-1 
2. Barbui, T., Thiele, J., Gisslinger, H., Kvasnicka, H. M., Vannucchi, A. M., Guglielmelli, P., Orazi, A., & Tefferi, A. (2018). The 2016 WHO classification and diagnostic criteria for myeloproliferative neoplasms: document summary and in-depth discussion. Blood Cancer J, 8(2), 15. https://doi.org/10.1038/s41408-018-0054-y 
3. Centogene. (2022). Myeloid Tumor Panel. https://www.centogene.com/diagnostics/our-tests/somatic-mutation-testing/somatic-mutation-testing-for-myeloid-tumors 
4. Coates, T. D. (2022, May 05, 2022). Approach to the patient with neutrophilia. https://www.uptodate.com/contents/approach-to-the-patient-with-neutrophilia
5. Cross, N. C. P., Godfrey, A. L., Cargo, C., Garg, M., Mead, A. J., & Paper, A. B. S. f. H. G. P. (2021). The use of genetic tests to diagnose and manage patients with myeloproliferative and myeloproliferative/myelodysplastic neoplasms, and related disorders. British Journal of Haematology, 195(3), 338-351. https://doi.org/https://doi.org/10.1111/bjh.17766 
6. EASL. (2016). EASL Clinical Practice Guidelines: Vascular diseases of the liver. J Hepatol, 64(1), 179-202. https://doi.org/10.1016/j.jhep.2015.07.040 
7. Faisal, M., Stark, H., Busche, G., Schlue, J., Teiken, K., Kreipe, H. H., Lehmann, U., & Bartels, S. (2019). Comprehensive mutation profiling and mRNA expression analysis in atypical chronic myeloid leukemia in comparison with chronic myelomonocytic leukemia. Cancer Med. https://doi.org/10.1002/cam4.1946 
8. FDA. (2017a).  https://www.accessdata.fda.gov/cdrh_docs/pdf17/K172287.pdf
9. FDA. (2017b). Approved Drugs - Ipsogen JAK2 RGQ PCR Kit [WebContent]. https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm551474.htm 
10. Grinfeld, J., Nangalia, J., & Green, A. R. (2017). Molecular determinants of pathogenesis and clinical phenotype in myeloproliferative neoplasms. Haematologica, 102(1), 7-17. https://doi.org/10.3324/haematol.2014.113845 
11. Harrison, C. N., Butt, N., Campbell, P., Conneally, E., Drummond, M., Green, A. R., Murrin, R., Radia, D. H., Mead, A., Reilly, J. T., Cross, N. C. P., & McMullin, M. F. (2014). Modification of British Committee for Standards in Haematology diagnostic criteria for essential thrombocythaemia. British Journal of Haematology, 167(3), 421-423. https://doi.org/10.1111/bjh.12986 
12. Hussein, K., Granot, G., Shpilberg, O., & Kreipe, H. (2013). Clinical utility gene card for: familial polycythaemia vera. Eur J Hum Genet, 21(6). https://doi.org/10.1038/ejhg.2012.216 
13. labcorp. (2023). IntelliGEN® Myeloid. https://oncology.labcorp.com/cancer-care-team/test-menu/intelligen-myeloid 
14. Maxson, J. E., Gotlib, J., Pollyea, D. A., Fleischman, A. G., Agarwal, A., Eide, C. A., Bottomly, D., Wilmot, B., McWeeney, S. K., Tognon, C. E., Pond, J. B., Collins, R. H., Goueli, B., Oh, S. T., Deininger, M. W., Chang, B. H., Loriaux, M. M., Druker, B. J., & Tyner, J. W. (2013). Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML. N Engl J Med, 368(19), 1781-1790. https://doi.org/10.1056/NEJMoa1214514 
15. McMullin, M. F., Harrison, C. N., Ali, S., Cargo, C., Chen, F., Ewing, J., Garg, M., Godfrey, A., S, S. K., McLornan, D. P., Nangalia, J., Sekhar, M., Wadelin, F., Mead, A. J., & the, B. S. H. C. (2019). A guideline for the diagnosis and management of polycythaemia vera. A British Society for Haematology Guideline. British Journal of Haematology, 184(2), 176-191. https://doi.org/10.1111/bjh.15648 
16. NCCN. (2019). NCCN Clinical Practice Guidelines in Oncology; Myeloproliferative Neoplasms v3.2019 https://www.nccn.org/professionals/physician_gls/pdf/mpn.pdf 
17. NCCN. (2022). NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines (R)): Myeloproliferative Neoplasms v3.2022. NCCN. Retrieved 02/07/23 from https://www.nccn.org/professionals/physician_gls/pdf/mpn.pdf
18. NeoGenomics. (2022a). MPN JAK2 V617F with Sequential Reflex to JAK2 Exon 12-13, CALR, and MPL. https://neogenomics.com/test-menu/mpn-jak2-v617f-sequential-reflex-jak2-exon-12-13-calr-and-mpl 
19. NeoGenomics. (2022b). NeoTYPE® Myeloid Disorders Profile. https://neogenomics.com/test-menu/neotyper-myeloid-disorders-profile 
20. Ojeda, M. J., Bragós, I. M., Calvo, K. L., Williams, G. M., Carbonell, M. M., & Pratti, A. F. (2018). CALR, JAK2 and MPL mutation status in Argentinean patients with BCR-ABL1- negative myeloproliferative neoplasms. Hematology, 23(4), 208-211. https://doi.org/https://doi.org/10.1080/10245332.2017.1385891 
21. Poluben, L., Puligandla, M., Neuberg, D., Bryke, C. R., Hsu, Y., Shumeiko, O., Yuan, X., Voznesensky, O., Pihan, G., Adam, M., Fraenkel, E., Rasnic, R., Linial, M., Klymenko, S., Balk, S. P., & Fraenkel, P. G. (2019). Characteristics of myeloproliferative neoplasms in patients exposed to ionizing radiation following the Chernobyl nuclear accident. Am J Hematol, 94(1), 62-73. https://doi.org/10.1002/ajh.25307 
22. Quest_Diagnostics. (2020). LeukoVantage® Myeloid Neoplasm Mutation Panels. https://www.questdiagnostics.com/healthcare-professionals/clinical-education-center/faq/faq208 
23. Rampal, R., Al-Shahrour, F., Abdel-Wahab, O., Patel, J. P., Brunel, J. P., Mermel, C. H., Bass, A. J., Pretz, J., Ahn, J., Hricik, T., Kilpivaara, O., Wadleigh, M., Busque, L., Gilliland, D. G., Golub, T. R., Ebert, B. L., & Levine, R. L. (2014). Integrated genomic analysis illustrates the central role of JAK-STAT pathway activation in myeloproliferative neoplasm pathogenesis. Blood, 123(22), e123-133. https://doi.org/10.1182/blood-2014-02-554634 
24. Rosenthal, S. H., Gerasimova, A., Ma, C., Li, H. R., Grupe, A., Chong, H., Acab, A., Smolgovsky, A., Owen, R., Elzinga, C., Chen, R., Sugganth, D., Freitas, T., Graham, J., Champion, K., Bhattacharya, A., Racke, F., & Lacbawan, F. (2021). Analytical validation and performance characteristics of a 48-gene next-generation sequencing panel for detecting potentially actionable genomic alterations in myeloid neoplasms. PLoS One, 16(4), e0243683. https://doi.org/10.1371/journal.pone.0243683 
25. Rumi, E., & Cazzola, M. (2017). Diagnosis, risk stratification, and response evaluation in classical myeloproliferative neoplasms. Blood, 129(6), 680-692. https://doi.org/10.1182/blood-2016-10-695957 
26. Silvennoinen, O., & Hubbard, S. R. (2015). Molecular insights into regulation of JAK2 in myeloproliferative neoplasms. Blood, 125(22), 3388-3392. https://doi.org/10.1182/blood-2015-01-621110 
27. Tefferi, A. (2022a, 09/13/2022). Clinical manifestations and diagnosis of primary myelofibrosis. https://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-primary-myelofibrosis
28. Tefferi, A. (2022b, 09/13/2022). Overview of the myeloproliferative neoplasms. https://www.uptodate.com/contents/overview-of-the-myeloproliferative-neoplasms
29. Tefferi, A. (2023a, 01/03/2023). Clinical manifestations and diagnosis of polycythemia vera. https://www.uptodate.com/contents/clinical-manifestations-and-diagnosis-of-polycythemia-vera
30. Tefferi, A. (2023b, 01/09/2023). Clinical manifestations, pathogenesis, and diagnosis of essential thrombocythemia. https://www.uptodate.com/contents/diagnosis-and-clinical-manifestations-of-essential-thrombocythemia
31. Tefferi, A., Thiele, J., Vannucchi, A. M., & Barbui, T. (2014). An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms. Leukemia, 28(7), 1407-1413. https://doi.org/10.1038/leu.2014.35 
32. Titmarsh, G. J., Duncombe, A. S., McMullin, M. F., O'Rorke, M., Mesa, R., De Vocht, F., Horan, S., Fritschi, L., Clarke, M., & Anderson, L. A. (2014). How common are myeloproliferative neoplasms? A systematic review and meta-analysis. Am J Hematol, 89(6), 581-587. https://www.ncbi.nlm.nih.gov/pubmed/24971434 
33. Vainchenker, W., & Kralovics, R. (2017). Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms. Blood, 129(6), 667-679. https://doi.org/10.1182/blood-2016-10-695940 
34. Vannucchi, A. M., on behalf of the, E. G. C., Barbui, T., on behalf of the, E. G. C., Cervantes, F., on behalf of the, E. G. C., Harrison, C., on behalf of the, E. G. C., Kiladjian, J. J., on behalf of the, E. G. C., Kröger, N., on behalf of the, E. G. C., Thiele, J., on behalf of the, E. G. C., Buske, C., & on behalf of the, E. G. C. (2015). Philadelphia chromosome-negative chronic myeloproliferative neoplasms: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†. Annals of Oncology, 26(suppl_5), v85-v99. https://doi.org/10.1093/annonc/mdv203

Coding Section

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, Blue Cross Blue Shield Association technology assessment program (TEC) and other nonaffiliated technology evaluation centers, reference to federal regulations, other plan medical policies, and accredited national guidelines.

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