Introduction

The myeloproliferative neoplasms (MPN) are cancerous proliferations of cells of the bone marrow and blood involving, to varying degrees, progenitors of white blood cells, red blood cells, and platelets. The MPNs span eight disease categories in the 2008 World Health Organization (WHO) classification of hematopoietic neoplasms. MPNs are generally distinguished from acute myeloid leukemia by the presence of fewer than 20% myeloblasts in the blood and bone marrow, and they are generally distinguished from other non-acute myeloid neoplasms by a lack of morphologic dysplasia, and by a pattern of “effective” hematopoiesis, either in the form of increased peripheral blood cell counts, splenomegaly, or both. This is as opposed to the pattern of “ineffective” hematopoiesis (cellular bone marrow but decreased peripheral cell counts) and morphologic dysplasia more typical of the myelodysplastic syndromes (MDS). Three of the major MPN categories—polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF)—are associated to varying degrees with a mutation in the Janus Kinase 2 (JAK2) gene.

As a result, assessment for JAK2 mutation status has become routine in the workup of patients with persistently elevated peripheral blood counts, organomegaly, unexplained thrombosis, or other signs suggestive of MPN. In addition, a minor subset of disorders classified by WHO in the category of myeloproliferative/myelodysplastic neoplasms (neoplasms that show overlap features of both MPNs and myelodysplastic syndromes) appear to be associated with JAK2 mutation.

The Janus kinases comprise a family of proteins named for the Roman god of transitions — a two-faced entity that was said to simultaneously face the past and the future — so named because the JAK proteins contain two principal binding domains, one that enhances and one that inhibits kinase activity. The JAK proteins are major components of the JAK-STAT pathway of cellular proliferation, and abnormalities in the JAK2 gene are thought to drive the proliferative process in JAK2-associated myeloid neoplasms. A substantial majority of JAK2-associated neoplasms harbor the JAK2 V617F mutation, in which a point mutation in the DNA code at exon 14 results in the substitution of a phenylalanine residue (F) for the normal valine (V) residue, resulting in gain of JAK2 function and enhancement of proliferative activity. A small percentage of JAK2-associated neoplasms harbor mutations other than the V617F; the most common of these alternative mutations occurs in exon 12 of the JAK2 gene.

All MPN are thought to represent hematopoietic stem cell neoplasms. In other words, neoplastic proliferation is thought to take place at the level of a pleuripotent stem cell that subsequently differentiates into multiple specific hematopoietic lineages (erythroid, megakaryocytic, granulocytic, monocytic). Before the discovery of associated gene fusions and mutations, the classification of a given MPN historically was based on the predominant pattern of differentiation of the neoplastic stem cell. Predominantly neutrophilic differentiation characterized chronic myelogenous leukemia (CML) (now known to be specifically linked to the Philadelphia chromosome / BCR-ABL gene fusion and not generally included in the JAK2 associated neoplasms); predominantly erythroid differentiation characterized polycythemia vera (PV); and predominantly megakaryocytic/ platelet differentiation characterized essential thrombocythemia (ET). Primary myelofibrosis (PMF) (historically termed myelofibrosis with myeloid metaplasia, agnogenic myeloid metaplasia, or chronic idiopathic myelofibrosis) was marked by an expansion of multiple cell lineages, often most prominently megakaryocytic, in which the megakaryocytic component stimulated marked marrow fibrosis through secretion of platelet-derived growth factors, and resulted in prominent foci of extramedullary neoplastic hematopoiesis (most notably in the spleen).

Although some hematologic malignancies are defined categorically by the presence of a specific molecular genetic abnormality (such as the PML-RARA fusion in acute promyelocytic leukemia), the current consensus is that JAK2 mutation-associated MPN should still be classified according to clinicopathologic features and not considered variants of a single disorder. A brief discussion of the four major JAK2 mutation-associated disease categories follows.

Polycythemia Vera

The hallmark of polycythemia vera (PV) is the neoplastic overproduction of red blood cells, resulting in polycythemia (by WHO criteria a persistent whole blood hemoglobin in excess of 18.5 g/dL in men, or 16.5 g/dL in women). However, bone marrow examination in PV patients typically shows a neoplastic expansion of all hematopoietic lineages (panmyelosis), rendering some morphologic similarities with other MPNs, particularly primary myelofibrosis (PMF).

Current WHO diagnostic criteria for a diagnosis of PV are summarized in Table 1. The findings of a persistently elevated whole blood hemoglobin level (greater than 18.5 g/dL in men, or greater than 16.5 g/dL in women), a documented JAK2 mutation, and serum erythropoietin below reference range are sufficient to diagnose PV by WHO criteria. However, a diagnosis can still be made in the absence of some of these features if other combinations of criteria are met (see Table 1).

Table 1. World Health Organization Criteria for a Diagnosis of Polycythemia Vera (Reproduced from Thiele et al, WHO, 2008). Diagnosis requires both major criteria and one minor criterion, OR the first major criterion combined with two minor criteria.

Major Criteria	Hemoglobin >18.5 g/dL in men, 16.5 g/dL in women, or other evidence of increased red cell volume*
Presence of JAK2 V617F or other functionally similar mutation such as JAK2 exon 12 mutation.
Minor Criteria	Bone marrow biopsy showing hypercellularity for age with trilineage growth (panmyelosis) with prominent erythroid, granulocytic, and megakaryocytic proliferation
Serum erythropoietin level below the reference range for normal
Endogenous erythroid colony formation in vitro

*Hemoglobin or hematocrit >99th percentile of method-specific reference range for age, sex, altitude of residence, or hemoglobin >17 g/dL in men, 15 g/dL in women if associated with a documented and sustained increase of at least 2 g/dL: from an individual’s baseline value that cannot be attributed to correction of iron deficiency, or elevated red cell mass >25% above mean normal predicted value.

Among the different MPNs, PV has the strongest association with JAK2 mutation. Approximately ninety-five percent of cases of PV are said to harbor a JAK2 V617F mutation. However, when variant mutations (particularly the exon 12 mutation) are included, the prevalence of JAK2 mutation in PV likely exceeds 99%. Therefore, a diagnosis of PV in the absence of some form of JAK2 mutation should generally be approached with great caution.

There are many non-neoplastic (secondary) causes for polycythemia, and if a JAK2 mutation cannot be documented in a patient with persistent polycythemia, then these secondary causes should be considered. A list of potential causes of secondary polycythemia is provided in Table 2.

Table 2. Potential Causes of Secondary Polycythemia

Chronic hypoxia (cyanotic heart disease, hypoxic lung disease, sleep apnea, etc)

High oxygen-affinity hemoglobinopathy

Anabolic steroids

Ectopic erythropoietin production (e.g. from a neoplasm)

Familial abnormalities in erythropoietin feedback mechanism (e.g. “Chuvash” polycythemia)

In general, PV is an indolent disorder, with median survival typically reported as exceeding 10 years. There is some risk of transformation to acute leukemia, but in the absence of previous cytotoxic chemotherapy this risk is small (2 to 3% according to WHO). There is a risk for development of post-polycythemic myelofibrosis, and this risk increases with time, occurring in up to 50% of patients who survive at least 20 years with the disease. The onset of post-polycythemic myelofibrosis is often heralded by the development of peripheral cytopenias, and in some cases post-polycythemic myelofibrosis may be essentially identical to primary myelofibrosis clinically and histologically.

Essential Thrombocythemia

Essential thrombocythemia is marked by a persistent increase in peripheral blood platelet count, generally with normal leukocyte and red cell counts. The bone marrow in essential thrombocythemia is generally either normocellular or only slightly hypercellular, and morphologic abnormalities are generally limited to subtle increases and subtle clustering of megakaryocytes. Megakaryocytes may also show complex morphologic features including hyperlobation. Marked marrow hypercellularity, or extensive or cohesive clustering of megakaryocytes in the bone marrow should raise suspicion for a different MPN, particularly the cellular phase of PMF (see below).

In contrast to PV, JAK2 mutation is generally documented in only about 50% of cases of ET. Since the peripheral blood platelet count may be elevated in a number of reactive or inflammatory conditions, and since there is substantial overlap in the magnitude of the platelet count between reactive thrombocytosis and ET, the diagnosis in JAK2-negative cases often involves the exclusion of potential secondary causes for thrombocytosis. In addition, since many MPNs may present initially with thrombocytosis, diagnostic criteria for ET include the exclusion of other WHO-defined MPNs. Finally, since PV may present initially with thrombocytosis, and since secondary iron deficiency may mask polycythemia, the diagnosis of ET in patients with evidence of iron deficiency (such as decreased serum ferritin levels) requires the failure of iron therapy to return hemoglobin level to PV range. The WHO criteria for the diagnosis of ET are summarized in Table 3.

Table 3. Summary of World Health Organization Diagnostic Criteria for Essential Thrombocythemia (Reproduced from Thiele et al, WHO, 2008). Please see text for additional explanatory notes.

Sustained platelet count >= 450 x 109/L (Sustained during work-up process)

Bone marrow biopsy specimen showing proliferation mainly of the megakaryocytic lineage with increased numbers of enlarged, mature megakaryocytes. No significant increase or left-shift of neutrophil granulopoiesis or erythropoiesis

Not meeting WHO criteria for polycythemia vera, primary myelofibrosis, BCR-ABL1 positive chronic myelogenous leukemia, or myelodysplastic syndrome or other myeloid neoplasm

Demonstration of JAK2 V617F or other clonal marker, or in the absence of JAK2 V617F, no evidence for reactive thrombocytosis

When strictly defined by WHO criteria, ET is an indolent disease, with some studies showing comparable survival to age-matched controls. Transformation to acute leukemia is rare, and while the WHO includes a category of post-ET myelofibrosis, the development of marrow fibrosis in a patient with an established diagnosis of ET should also raise the possibility of primary myelofibrosis presenting initially as isolated thrombocytosis (see below).

Primary Myelofibrosis

Primary myelofibrosis (PMF) historically has gone by several different names, including myelofibrosis with myeloid metaplasia (MMM), agnogenic myeloid metaplasia (AMM), and chronic idiopathic myelofibrosis (CIMF). Typical fibroticphase PMF is associated with a distinct array of peripheral blood and bone marrow findings. The peripheral blood
typically shows leukoerythroblastic changes—that is, the combined presence of nucleated red blood cells and immature neutrophil precursors in the blood. Teardrop-shaped red blood cells (dacryocytes) are also typically seen. These changes have been described as correlating with marrow fibrosis, but may actually be a reflection of the extramedullary hematopoiesis (particularly splenic hematopoiesis) typical of fibrotic phase PMF. Fibrotic phase PMF is marked by the development of reticulin fibrosis, or in more advanced disease a more mature collagen fibrosis displacing the hematopoietic marrow space. The fibrosis is thought to be caused by the secretion of growth factors from the neoplastic megakaryocytes of this disorder. In its prefibrotic phase, PMF is marked by marrow hypercellularity due to proliferation of granulocytic precursors and megakaryocytes, often with very prominent and cohesive megakaryocyte clustering. The presence of megakaryocytes or other marrow precursor cells in bone marrow sinuses (intrasinusoidal hematopoiesis) also may be seen in PMF, and may provide a clue to diagnosis. Splenomegaly is a common clinical sign at presentation.

As is the case with ET, only about 50% of cases of PMF are associated with JAK2 mutation. Another 10% of cases are associated with activating mutations of the thrombopoietin receptor gene MPL. In addition, a number of other, much less prevalent, gene mutations have been described in associated with JAK2-negative PMF.

True fibrotic phase PMF carries a median survival duration of approximately five years. However, a number of patients present in prefibrotic phase with more prolonged survival duration. The clinical and histopathologic findings of prefibrotic PMF may be subtle and nonspecific. In some cases, patients present with isolated thrombocytosis mimicking ET,
but with certain findings atypical for ET such as marked splenomegaly, marked marrow hypercellularity, extensive megakaryocyte proliferation or cohesive or extensive megakaryocyte clustering in the marrow. There has been increased focus in recent years on the distinction between ET and prefibrotic PMF based on these types of findings. A summary of WHO criteria for a diagnosis of PMF is provided in table 4.

Table 4. Summary of World Health Organization Diagnostic Criteria for Primary Myelofibrosis (Reproduced from Thiele et al, WHO, 2008). Diagnosis requires all three major criteria and two minor criteria.

Major Criteria	Presence of megakaryocyte proliferation and atypia, usually accompanied by either reticulin and/or collagen fibrosis, OR, in the absence of significant reticulin fibrosis, the megakaryocyte changes must be accompanied by an increased bone marrow cellularity characterized by granulocyte proliferation and often decreased erythropoiesis (i.e. prefibrotic or cellular phase disease).
Not meeting WHO criteria for polycythemnia vera, BCR-ABl1 positive chronic myelogenous leukemia, myelodysplastic syndrome, or other myeloid neoplasms.
Demonstration of JAK2 V617F or other clonal marker (e.g. MPL W515K/L)
Minor Criteria	Leukoerythroblastosis
Increased serum lactate dehydrogenase (LDH) level
Anemia
Splenomegaly

PMF may be mimicked by other conditions as well. As noted above, post polycythemic myelofibrosis in patients with established PV may be indistinguishable from PMF except for the known history of antecendent PV. A number of conditions may lead to secondary fibrosis of the bone marrow, not driven by a primary myeloid neoplasm. Such
causes include metastatic carcinoma to bone with extensive fibrotic reaction, various bone disorders including hyperparathyroidism, and autoimmune myelofibrosis. Autoimmune-associated fibrosis may be extensive in the marrow, but by definition should be accompanied by the detection of some form of autoantibody on serologic evaluation, and should not be accompanied by the other typical morphologic features of PMF such as intrasinusoidal hematopoiesis or abnormal megakaryocyte clustering in the marrow, or teardrop poikilocytosis in the blood.

Other JAK2-Associated Myeloid Neoplasms

Occasionally, myeloid neoplasms not classifiable as PV, ET, or PMF by WHO criteria may harbor the JAK2 V617F mutation. Chronic myelomonocytic leukemia (CMML), a disorder with features that overlap between myelodysplastic syndromes and myeloproliferative neoplasms, has been reported to harbor JAK2 mutation in a small subset of cases. There have been sporadic reports of JAK2 mutations in certain myelodysplastic syndromes and even in acute myeloid leukemia. A provisional WHO diagnostic entity known as “refractory anemia with ring sideroblasts and thrombocytosis (RARS-T)” also has been associated with JAK2 mutation in roughly half of cases (a similar JAK2 mutation prevalence as seen in ET and PMF). RARS-T shows features similar either to ET or cellular phase PMF, but with the distinct finding of ring sideroblasts in the bone marrow. Ring sideroblasts are a light microscopic manifestation of abnormal iron sequestration within mitochondria of red blood cell precursors, and are often associated with certain myelodysplastic syndromes or toxic/ metabolic abnormalities of bone marrow. RARS-T is now considered among the categories of JAK2 mutation-associated myeloid neoplasms.

JAK2 As a Target for Therapy

In 2011, the JAK2 inhibitor ruxolitinib was approved by the FDA for the treatment of intermediate or high risk PMF, as well as post PV myelofibrosis and post ET myelofibrosis. Approval was based upon clinical trials data that showed significant reductions in symptoms and significant reduction in splenomegaly in patients treated with this drug. Since initial approval, a large, double-blind, placebo-controlled clinical trial also demonstrated a statistically significant improvement in median survival for patients treated with ruxolitinib relative to patients treated with placebo. JAK2 inhibitor therapy will no doubt play a key role in the treatment of MPNs going forward.