Thrombocytosis: Essential Thrombocythemia and Reactive Causes

Pathogenesis of Essential Thrombocythemia

In 1981 ET was first recognized as a disease that arises from clonal platelet expansion by pluripotent stem cells. This was accomplished by using X-chromosome–linked gene probes, such as those used for glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase, and hypoxanthine phosphoribosyltransferase, and provided a powerful tool to study large populations of patients with ET.

TPO, as the primary regulator of platelet production, stimulates the growth and differentiation of megakaryocyte progenitor cells in vitro and in vivo. TPO binds to c-MPL receptors (the product of the c-MPL proto-oncogene) on the platelet membrane surface and is subsequently internalized and degraded. Plasma TPO levels normally are regulated by total platelet and megakaryocyte mass and are highly elevated in patients with aplastic anemia. It is interesting to note that TPO levels are normal or slightly elevated in ET, despite an expanded megakaryocyte and platelet mass.

Dysregulation of the TPO c-MPL system in ET is suggested. In fact, patients with ET have markedly decreased expression of c-MPL protein on their platelet membranes (and decreased messenger RNA expression), which results in reduced TPO-binding capacity, impaired uptake and catabolism of TPO, and decreased clearance of TPO from the circulation. These findings explain the normal or slightly increased plasma TPO levels that occur in ET and suggested early on that megakaryocytes in ET may be hypersensitive to the stimulatory effects of TPO on production of platelets in vivo. Reduced expression of c-MPL cannot be used as a reliable diagnostic marker to distinguish ET from secondary causes of elevated platelet counts, because TPO levels may also be elevated in both reactive and clonal thrombocytosis.

Since the discovery of the JAK2 V617F gene mutation, and subsequently the MPL and CALR gene mutations, and their purported roles in the pathogenesis of the MPNs, clinicians have come to appreciate that ET is pathogenetically heterogeneous and that clonal thrombopoiesis is frequent ( Fig. 19.2 ). It is important to understand that these gene mutations, although mutually exclusive, are not causal. No consistent gene mutation or chromosomal defect or causal gene association for ET has been identified to date.

All MPNs are clonal disorders with an initial “hit” in specific hematopoietic lineage stem cells, resulting in excessive production of that lineage cell line and reflecting either hypersensitivity to cytokines and growth factors or autonomy from normal cytokine regulation. Whichever mechanism prevails, myeloproliferation results from the absence of normal feedback regulation.

The JAK2 V617F mutation on exon 14 is a “gain-of-function” mutation resulting in hypersensitivity or even independence of intracellular JAK-STAT pathway signaling to external stimulation of the MPL, EPO, and G-CSF receptors by circulating cytokines and growth factors (see Fig. 19.2 ). This remains the most frequent molecular diagnostic finding in ET, because approximately 50% to 55% of ET patients possess the JAK2 V617F mutation.

More recently, two additional mutually exclusive types of driver mutations have been implicated in the clonal expansion seen in ET patients: MPL and CALR mutations. The effect of these mutations is restricted to MPL activation and therefore is prevalent in ET and post-ET MF, but almost never seen in PV. There have been several individual MPL mutations identified, although the most frequent are MPL 515L and 515K. All lead to constitutive activation of the MPL receptor in the absence of TPO or enhance stability of receptor dimerization, leading to hypersensitive JAK2 activation and subsequently to platelet production. These mutations occur with less frequency and are found in only 2% to 3% of patients with ET. In contrast, CALR is neither an external receptor nor an intracellular signaling molecule, but serves as a chaperone of proteins through the endoplasmic reticulum. Mutated CALR facilitates both heightened trafficking and binding to MPL, causing hyperresponsiveness to TPO and activation of STAT-mediated platelet production (see Fig. 19.2 ). Although several CALR mutations have been identified (some unimplicated in ET pathogenesis), the two most commonly associated with ET are a 52 base pair deletion, called type 1, and a 5 base pair insertion, called type 2. Taken together, these mutations are detected in 20% to 25% of patients with ET, where both types are predominant (with a slight predilection of type 1). Each of the CALR mutations conveys its individual implication for survival prognosis and thrombotic risk. It is not yet known if each mutation responds differently to modality of treatment.

Criteria for the Diagnosis of Essential Thrombocythemia

In 1923 Minot and Buckman recognized the occurrence of multiple hemorrhagic episodes to be associated with very high platelet counts in patients with MPNs. In 1934 Epstein and Goedel, two Austrian hematologists, introduced the term hemorrhagic thrombocythemia for the association of recurrent hemorrhages with extreme thrombocytosis ( vasculärer Schrumpfmilz ) that had persisted for longer than 10 years. In 1960 Gunz refined the definition of hemorrhagic thrombocythemia into a clinical syndrome characterized by recurrent spontaneous hemorrhage, often preceded by thrombosis; extremely high platelet counts, usually in excess of 1,000,000/µL; frequent association with splenomegaly and hypochromic anemia; and a tendency to develop erythrocytosis (PV) between hemorrhagic episodes. In 1975 this entity was designated ET by the Polycythemia Vera Study Group (PVSG), which at that time established the diagnostic criteria for ET as (1) platelet counts in excess of 1,000,000/µL, (2) marked megakaryocytic hyperplasia on bone marrow aspiration, and (3) absence of PV, no significant MF, and Ph ¹ chromosome negativity. The PVSG set of parameters was intended to tighten clinical criteria for patient participation in research studies and facilitated the ability to discriminate RT from ET and other clonal causes of thrombocytosis (CML, PV, and the hypercellular stage of primary MF and MDS).

Since the late 1970s and early 1980s a number of clinical trials recognized the presence of a distinct cohort of symptomatic ET patients, who experienced microvascular peripheral disorders (erythromelalgia) or cerebral arterial complications (transient ischemic attacks [TIAs], migraines, etc.) when their platelet counts ranged between 400,000 and 1,000,000/µL. This prompted the PVSG to lower the platelet count criterion for the diagnosis of ET to an arbitrary minimum of 600,000/µL. In addition, the PVSG criteria consider the presence of a normal serum ferritin level concurrent with normal red blood cell (RBC) mean corpuscular volume to be sufficient evidence to exclude both RT caused by iron deficiency and PV masked by iron deficiency as possible causes of the thrombocytosis. Subsequently, the World Health Organization (WHO) refined the platelet count indicative of ET to be a sustained value higher than 450,000/µL, based on well-conducted retrospective analyses of data for ET patients, which revealed that early ET may be characterized by platelet counts ranging from slightly greater than normal to 600,000/µL ( Box 19.1 ).

In most studies the average platelet count at the time of diagnosis of ET has hovered around 1,000,000/µL. The hematocrit has usually been normal unless the clinical course has been complicated by bleeding or iron deficiency. Mild leukocytosis (range 10,000 to 20,000/µL) is commonly seen and may be associated with myeloid immaturity and a left shift in the differential count. Basophilia and/or eosinophilia may be present, as in other MPNs. Serum potassium levels may be spuriously elevated because of the very high platelet count observed in the presence of normal renal function (pseudohyperkalemia; i.e., increased serum potassium levels but normal plasma potassium levels). This pseudohyperkalemia is explained by the release of potassium from platelets during in vitro coagulation and clot retraction. Measurement of plasma potassium level provides a more accurate assessment. One-fourth of patients may have elevated levels of serum lactate dehydrogenase (LDH) and uric acid. The leukocyte alkaline phosphatase level is normal in most patients, although abnormally increased or reduced levels are not uncommon.

Currently, the diagnosis of the MPNs is facilitated by a stepwise molecular analysis of the mutually exclusive JAK2 V617F, CALR, and MPL mutations. Given the aforementioned prevalence of these genetic drivers, it is estimated that 75% to 80% of ET patients will test positive for an activating mutation, whereas the remaining 20% to 25% are termed “triple-negative” ET. When clonal markers are not present, ET becomes a diagnosis of exclusion after RT, PV, MF, CML, MDS, and other myeloid neoplasms are ruled out as a cause of the thrombocytosis.

A prominent requirement to establish the diagnosis of ET, according to WHO criteria ( Table 19.4 and Box 19.1 ), is the finding on bone marrow aspirate or biopsy specimen of megakaryocytic hyperplasia with loosely clustered large and giant megakaryocytes with mature cytoplasm and hyperploid multilobulated nuclei in the context of relatively normal myelopoiesis and erythropoiesis. In the overall population of patients with ET, bone marrow cellularity is normal in 52% and slightly increased in the remainder, primarily because of erythroid hyperplasia. Increased reticulin fibrosis is not a prominent feature of the bone marrows of ET patients but is a key component of the bone marrows of patients with chronic idiopathic (primary) myelofibrosis (CIMF) ( Table 19.5 ) and, to a lesser extent, of those with PV.

In 2001 the WHO developed bone marrow criteria to differentiate between true ET, early PV mimicking ET, and the extreme thrombocytosis associated with prefibrotic CIMF (CIMF-0) (see Table 19.4 ). Dense clusters of atypical immature megakaryocytes containing irregular cloudlike nuclei were present in CIMF-0 but were almost never seen in ET and PV. Thus, when the WHO bone marrow criteria were applied retrospectively to 839 patients who had previously been diagnosed with ET according to the PVSG criteria (platelet count of >600,000/µL), true ET was corroborated in 21%, CIMF-0 in 27%, CIMF-1 in 40%, and CIMF-2 in 13%.

The 2016 revision to the WHO classification of ET changed only slightly from the 2008 version with the addition of CALR mutation testing (see Box 19.1 , Table 19.4 ). The criteria continue to underscore the importance of bone marrow evaluation even in the presence of a diagnostic mutation, to adequately assess for prefibrotic MF. The diagnostic dilemma signified by suspicion of ET in the absence of a driver mutation highlights the continued role of a comprehensive bone marrow evaluation in the diagnostic workup and fuels the continued search for additional gene mutations, which could be used to distinguish ET from RT and other disorders.

RT is more specifically excluded when polymerase chain reaction (PCR) assays detect the bcr/abl gene rearrangement, even when the Ph ¹ chromosome is absent or when cytogenetic studies detect the del(5q) mutation, which is associated with an MDS variant that often presents with marked thrombocytosis. There is recent evidence to suggest that gene expression profiling can be used to classify platelet phenotypes. Using a set of 11 genetic biomarkers for microarray profiling, Gnatenko and colleagues have demonstrated that RT, ET, and normal platelets are genetically distinct. In the future, this approach may increase the ability to distinguish among the causes of thrombocytosis and their clonal versus nonclonal origins.

The TET2 (ten-eleven transformation-2) and ASXL1 (Additional SeX combs–Like protein-1) gene mutations have also been examined as potential discriminators of ET from RT. They are found in very low frequency in ET (<3%), may precede or follow development of the JAK2 V617F mutation, and may be epigenetic regulators of cellular transformation.

Approximately 50% of patients with ET have increased expression of the polycythemia rubra vera 1 (PRV-1) gene. The PRV-1 gene is a member of the urokinase (UK)-type plasminogen activator receptor superfamily and is overexpressed in the granulocytes isolated from patients with PV and ET. This overexpression could also be a potential molecular marker that differentiates ET from RT.

Bone marrow and peripheral blood samples from approximately 50% of patients with ET show the ability to form spontaneous endogenous erythroid colonies (EECs) in vitro, which suggests that assays for the formation of EECs, and spontaneous endogenous megakaryocytic colonies (EMCs) may be useful in distinguishing between ET and RT. In one study, EEC positivity had a diagnostic sensitivity of 65% for ET and only one of 88 RT cases showed formation of EECs. In two other studies of patients with ET ( n = 204) and RT ( n = 59), the detection of EMCs was modest (sensitivity of 63% [ET] and 69% [RT], respectively), but the diagnostic specificity was 100% for ET. Although such in vitro assays are meaningful from a pathophysiologic perspective, they are difficult and expensive to perform on an individual basis and are reserved primarily for clinical research efforts. Potentially important is the observation that the results of in vitro testing for spontaneous EMC and EEC may have clinical significance as well as diagnostic significance. ET patients who tested positive for EECs and PRV-1 overexpression manifested a higher risk of microvascular and major thrombotic complications than ET patients who tested negative for spontaneous EEC formation and increased PRV-1 expression. ET with overexpression of PRV-1 may represent a pathobiologically distinct subcategory of ET associated with increased risk of development of thrombotic complications and eventual emergence of PV (see Table 19.4 ). The JAK2 V617F mutation has been strongly correlated with PRV-1 overexpression and the ability to form spontaneous EECs in all three subtypes of MPN.