Essential Thrombocythemia: A Review of Diagnostic and Pathologic Features

Historically, essential thrombocythemia (ET) has been called hemorrhagic thrombocythemia/thrombocytosis, primary thrombocythemia, and idiopathic thrombocythemia. The original term, hemorrhagic thrombocythemia, was described by Epstein and Goedel in Virchows Archiv für pathologische Anatomie und Physiologie und für klinische Medizin in 1934. The title of this German paper, "Hämorrhagische Thrombocythämie bei vascularer Schrumpfmilz," roughly translates as "Hemorrhagic thrombocythemia in a case of vascular (thrombosis-in-duced) splenic atrophy." This title beautifully describes the dual clinical manifestations of hemorrhage and thrombosis in a patient with thrombocythemia. In a 1951 Blood article, Dameshek speculated that ET belonged in the category of chronic myeloproliferative disorders (CMPDs) along with chronic myelogenous leukemia (CML), polycythemia vera (PV), and chronic idiopathic myelofibrosis (CIMF). The rare familial thrombocythemias are not included in the category of CMPDs, but they will be discussed briefly here.1-3

The definition and diagnostic criteria for ET have changed substantially during the last 30 years. For example, previously established diagnostic criteria for ET included a platelet count of =1000 × 10^sup 3^/µL, now revised to =600 × 10^sup 3^/µL (2600 × 10^sup 9^/L) in the newer accepted and published criteria. Splenomegaly, once a diagnostic criterion many years ago, is no longer required under the most recent 2001 World Health Organization (WHO) classification (Table 1); in fact, significant or progressive splenomegaly speaks against ET.1,4,5 Also now required for a diagnosis of ET is a bone marrow biopsy, which in the past some authors suggested was not valuable.6 Upon reevaluation of ET cases previously diagnosed by the PoIycythemia Vera Study Group criteria and subsequently reclassified according to WHO criteria, significant discrepancies have been found.7 Recently described cytogenetic abnormalities or molecular features characteristic of other well-defined CMPD or myelodysplastic syndromes (MDS), such as t(9;22) and its associated BCR/ABL fusion, del(5q), t(3;3)(q21;q26), and inv(3)(q21q26), must be absent for a definitive diagnosis of ET.1

EPIDEMIOLOGY

The concept and diagnostic criteria of ET have evolved to the point that what was once regarded as the least common CMPD now surpasses CML as the most common.1,4 Using the current WHO criteria, the annual incidence of ET is 1 to 2.5 per 100000 individuals. CML has now taken a close second place in frequency with an annual incidence of 1 to 2 per 100000 individuals.1 This apparent change in incidence can almost certainly be attributed to the increased and widespread use of routine complete blood count screening tests. In theory, the actual annual incidence of ET may be even higher than currently estimated by the WHO, because many patients may remain asymptomatic and thus never be diagnosed with ET unless seen by a physician for an unrelated illness. Currently, up to 50% of patients diagnosed with ET are first suspected to have the disease only when an elevated platelet count is found incidentally on routine complete blood count laboratory studies. In addition, the diagnosis of ET is highly reliant on clinicopathologic correlation and ultimately remains a diagnosis of exclusion after other reactive and neoplastic conditions have been ruled out as a cause of thrombocytosis. Therefore, the surgical pathologist or hematopathologist may not make the diagnosis of ET as often as one would expect because (1) not enough clinical data is available or (2) a bone marrow biopsy was not performed.

ET is primarily diagnosed in older (50-60-year-old) patients, with equal percentages of males and females. There also appears to be a second peak of patients diagnosed at around age 30, showing a preponderance of females.1 Racial differences have not been well studied, but it appears that ET, PV, and CIMF have a significantly lower incidence in the Mexican population compared to that of the United States as a whole.8 Several recent studies indicate that patients with ET may have shortened 5- and 10-year survival rates when compared to age-matched controls.9,10 However, other studies show that since patients are diagnosed at an older age, they have a near-normal life expectancy.1,11,12

CLINICAL FEATURES

The clinical manifestations of ET arise mostly from arterial and microvascular thrombotic events.13-15 Arterial thromboses are reported 3 times more often than venous thromboses. Venous events usually occur in the form of lower extremity deep vein thrombosis, portal and hepatic vein thrombosis, and pulmonary embolism. The most common sites for large arterial vessel thrombosis, in decreasing order of frequency, are the brain, heart, and extremities.13

Microvascular thrombi may result in erythromelalgia, headache, paraesthesia, digital ischemia, or necrosis.13,16,17 These microvascular phenomena in ET and PV can occur when platelet counts are as low as 400 × 10^sup 3^/µL. Reports suggest that the most common cause of secondary erythromelalgia is either ET or PV.18 The platelets in ET and PV are abnormally sensitive and aggregate at intermittent and unpredictable times, independent of platelet count, in the tiny blood vessels of the extremities (legs and hands most commonly), resulting in the manifestations of erythromelalgia, which typically include erythema, pain, and warmth.12,17 Erythromelalgia may also rarely affect the face.19

Extramedullary hematopoiesis can occur to a limited degree in the liver or spleen. Splenomegaly is seen in up to 20% to 50% of patients, although the enlargement is mild to moderate and not usually progressive. Hepatomegaly may be seen in 15% to 20% of patients, but significant extramedullary hematopoiesis in the liver is unusual and should raise the possibility of a diagnosis of CIMF or another CMPD.1,5,20

When the platelet count is greater than 1 to 1.5 million/mL, patients are at increased risk for hemorrhagic events.10,21 Bleeding, most commonly from the skin, mucous membranes, or gastrointestinal tract, is usually not severe unless the patient is on aspirin or other anticoagulants.5,13,22 The overall risk of bleeding and thrombosis is 0.33% per patient-year and 6.6% per patient-year in ET versus 0% and 1.2%, respectively, in the control population.13,14 Bleeding in many cases results from an acquired von Willebrand Syndrome (AvWS), which usually improves with platelet-lowering therapy.13,18,23

It has been reported that ET may rarely evolve into myelofibrosis or acute leukemia.1,24 However, this topic is controversial, as some investigators suggest that ET by definition should not have fibrosis and thus cases of ET that appear to evolve into myelofibrosis may actually represent the unrecognized cellular/prefibrotic stage of CIMF or PV.7 It is difficult to prove one way or the other, since no specific molecular, chromosomal, or biochemical marker has been identified to separate these cases. Another theoretical consideration is that ET could evolve into another higher-grade disease like CIMF, acute leukemia, or even MDS as additional DNA and biochemical changes and abnormalities accumulate. Regarding evolution to acute leukemia, it is uncommon (less than 5% of patients)1,25 and may occur sporadically or as a result of certain cytoreductive therapies, the latter being more common. The most common type of leukemia to occur in association with ET is acute myelogenous leukemia with M4 or M7 phenotype.26 It has been reported by many that previous cytoreductive therapy with hydroxyurea (HU) may slightly increase the risk for development of acute leukemia and myelodysplasia. There are also reported series of congenital heart disease patients and sickle cell patients who have been treated for years with HU and do not appear to have a significantly increased incidence of acute leukemia27,28; however, these findings are complicated by the possibility that these patients may die of other causes related to their disease (eg, acute chest syndrome) before they develop leukemia. Recent studies do appear to show that busulfan, HU, and radiophosphorus (^sup 32^P), alone or in combination, impart an increased risk for development of leukemia and myelodysplasia. Overall, the reports claim an incidence of acute leukemia varying from about 3% of patients treated with HU alone to up to 30% of patients when HU is preceded by busulfan.26,29-35 Anagrelide and interferon-a have not been shown to be leukemogenic.25,36

PATHOGENESIS

ET, as currently defined by the WHO, is a clonal proliferation of pluripotent stem cells with predominately megakaryocytic differentiation. However, recent studies using DNA and RNA X-chromosome inactivation patterns show that a substantial proportion of cases (up to 30% to 50%) diagnosed as ET have polyclonal hematopoiesis including megakaryocytopoiesis.5,13,24,30,37-39 Comparison of the polyclonal and monoclonal groups has shown an increased rate of thrombosis in the monoclonal group, with no significant differences identified in platelet count, age at diagnosis, or presence of splenomegaly. This raises the questions of whether ET is a heterogenous disease and whether our diagnostic skills and diagnostic criteria need further refinement.

Many abnormalities relating to platelets and other cell lines have been demonstrated in ET. Paradoxically, thrombopoietin levels in ET may be high or normal; this is in contrast to the decreased levels of erythropoietin in PV. The elevated thrombopoietin (TPO) levels in ET may be related to the abnormal TPO receptor (MPL) on the abnormal platelets and megakaryocytes in ET.20 Under normal circumstances, TPO binds to the MPL receptor in order to stimulate growth and proliferation. It is postulated that in ET, TPO does not bind properly to the defective or decreased MPL receptor expression,40 thereby leaving unbound TPO in the circulatory system. DNA structural abnormalities or mutations in the MPL receptor have not been identified,41 so it has been suggested that abnormalities in mRNA translation or posttranslational events may play a role.42 Increased MPL mRNA expression has been identified in the bone marrow cells of all CMPDs except for CML,43 while the MPL receptor has been found to be significantly decreased in ET.40,44,45 Another feature related to abnormal megakaryocyte growth and proliferation in ET is the finding that megakaryocytic and erythroid cell lines from these patients can grow in in vitro cultures autonomously without external growth factors, including thrombopoietin, from bone marrow progenitor cells.20,41 It has been suggested that ET patients with endogenous erythroid colony growth may have a higher rate of conversion to a clinicopathologic disease resembling PV.46

The platelets in ET show functional laboratory abnormalities as well.5,47-49 Overall, they show defective aggregation in 73% of patients. Specifically, they show abnormal response to epinephrine (58%), collagen (38%), and adenosine diphosphate (11%) in a variable number of assays using the Platelet Function Analyzer (PFA-100). Abnormal PFA aggregation studies, however, did not correlate with bleeding or bleeding time.47 The platelets may also show a decreased number of alpha-granules and, to a lesser degree, decreased dense bodies. Alpha-granules in normal platelets contain platelet-derived growth factor, platelet factor 4, factors V and XIII, and fibrinogen. Dense bodies contain serotonin, adenosine diphosphate, and calcium.50 Deficiency of intraplatelet von Willebrand factor (vWF) and fibrinogen is more frequently observed in ET and PV than in CML or CIMF.51 Patients with reactive thrombocytosis show normal platelet aggregation responses.52

Paradoxical bleeding in ET (hemorrhagic thrombocythemia) is associated with extremely high platelet counts (above 1000-1500 × 10^sup 3^/µL).10,21 Many of the cases of bleeding have been attributed to an acquired vWF deficiency or dysfunction known as AvWS. This corresponds to type II von Willebrand disease where there is loss of large vWF multimers.13 This phenomenon is not specific to ET and can be seen in reactive thrombocytosis; however, in reactive conditions patients are not prone to bleeding.53 The pathogenesis of AvWS in CMPDs is not completely understood, but it has been suggested to be the result of enhanced proteolysis of vWF multimers, presumably by the ADAMTS13 cleaving protease. The enhanced proteolysis theory assumes platelets are involved in increased proteolysis of vWF multimers, and exactly how this occurs in ET with AvWS has not been completely elucidated.13,23,54-57 Another theory explaining AvWS is that the large vWF multimers preferentially adsorb to the surface of platelets via membrane receptors.53

A series of recent studies have demonstrated increased polymorphonuclear leukocyte (PMN) activation in ET and PV compared to normal controls.13,58-60 PMN activation parameters, including CD11b, leukocyte alkaline phosphatase, cellular elastase, plasma elastase, and myeloperoxidase, have been found to be elevated in patients with ET and PV. In addition, PMN activation has been shown to correlate with plasma clotting and endothelial markers, suggesting a role for activated PMNs in the thrombotic events in ET and PV. PMNs contribute to clotting activation by release of intracellular granule contents. PMNs also bind platelets through increased CD11b expression on their activated cell membrane. Increased circulating PMN-platelet aggregates have been seen in PV and ET as well as in other prothrombotic states such as unstable angina, cardiopulmonary bypass, stroke, myocardial infarction, and hemodialysis.60,61 The PMN-platelet aggregates formed through CD11b/CD42b and CD11lb/CD62P interactions are increased in ET patients, and compared with PV patients, ET patients showed only increased CD11b/CD42b, suggesting differences in ET and PV neutrophilplatelet aggregates.60 Overall, though, the number of circulating neutrophil-platelet aggregates in both PV and ET are increased compared to other CMPDs and normal controis.58 These findings suggest a role for activated PMNs and platelets in the thrombotic events of ET.

CYTOGENETICS AND MOLECULAR MARKERS

Unlike in CML, no consistent, specific, or universal recurring cytogenetic abnormality or molecular marker has been identified in ET to date. However, numerous recent studies have identified a promising new molecular marker for ET and other non-CML CMPDs involving a mutation in the Janus kinase 2 gene (JAK2) located on chromosome 9p.46 JAK2 codes for a nonreceptor cytoplasmic proteintyrosine kinase in a variety of CMPDs.62-66 JAK2 is involved in signaling pathways required for hematopoiesis, including production of Bcl-2, interleukin (IL)-3, IL-5, erythropoietin, granulocyte-colony stimulating factor, and thrombopoietin.61 An acquired point mutation, G to T, leads to a valine to phenylalanine substitution at amino acid 617 of the JAK2 protein (JAK2^sup V617F^).46 The JAK2^sup 617F^ mutation results in constitutive activation of the tyrosine kinase in JAK2.64 The frequency of JAK2 mutations in CMPDs varies according to subtype but appears to occur least frequently in ET. The frequency of JAK2 mutations in the ET cases published to date ranges from 23% to 57%63,67-69 Additional JAK2 studies and standardized methods of testing for JAK2 are needed before this test can be considered for use in routine clinical practice. It should be pointed out that JAK2 mutations are not specific for the CMPDs and have been found uncommonly in other myeloid malignancies, including acute myelogenous leukemia and chronic myelomonocytic leukemia.70 JAK2 has not been found in lymphoid malignancies, including acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL).65

Various cytogenetic abnormalities have been identified in 5% of ET patients.71-73 Some authors have reported an increased frequency of trisomy 8 and trisomy 9 in ET, while others report no increased frequency of this cytogenetic abnormality.72-74 While recurring cytogenetic abnormalities in ET have not been established, some recurring findings have been identified in association with transformation to acute leukemia. Cytogenetic abnormalities seen with ET in association with acute myelogenous leukemia include t(2;17), t(3;17)(p24;q12), t(1;7), long-arm trisomy of chromosome 1, monosomy 7q, and deletion 17p. 33,75-79 Deletion 17p (site of p53 gene) has a high association with previous HU therapy. Pipobroman therapy is associated with long-arm trisomy of chromosome 1 and a monosomy 7q. Der(1;7)(q10;p10) has been associated with acute leukemic transformation in patients not treated with cytotoxic agents.77 Using comparative genomic hybridization, a relatively new molecular cytogenetic technique, a gain of 18p was seen in 1 of 8 patients with ET.71 By definition, there should not be t(9;22) as seen in CML or del 5q-, t(3;3)(q21;q26), or inv(3)(q21q26) as seen in myelodysplastic syndromes that may have thrombocytosis.1

Newer markers in ET are being investigated that may help define the disease and aid in diagnosis. A platelet-expressed gene (HSD17B3) encoding type 3 17-ß-hydroxysteroid dehydrogenase is selectively down-regulated in ET platelets, with reciprocal induction of the type 12 enzyme (HSD17B12). Proapoptotic genes such as BAX, BNIP3, and BNIP3L have been shown to be down-regulated in ET megakaryocytes.80 CD34-derived megakaryocytes from ET patients show up-regulation of antiapoptotic genes and the survival-enhancing gene SDF-1. In other experiments, ET megakaryocytes were more resistant to apoptosis when compared to healthy controls.46-80 Overexpression of polycythemia rubra vera-1 (PRV-1) mRNA in granulocytes has been described in PV, CIMF, and ET. 81-84 Overexpression of PRV-1, an allele associated with the CD177 receptor, may define a subset of patients who currently meet ET criteria but later evolve into PV.46

MORPHOLOGIC FINDINGS

The blood smear in ET shows marked thrombocytosis, often above 1000 × 10^sup 3^/µL and in some reported cases up to 14000 × 10^sup 3^/µL. The WHO uses >600 × 10^sup 3^/µL (=600 × 10^sup 9^/L) as the required sustained platelet count to make a diagnosis of ET The definition of "sustained" is not explicitly stated, although some authors use 2 platelet count tests showing >600 × 10^sup 3^/µL on at least 2 occasions and 2 months apart.85 The platelets may show marked anisocytosis, bizarre shapes, and hypogranulation. However, in some cases the platelets may have a normal shape and size. The white blood cell count is usually not significantly elevated, but may be mildly increased (20 × 10^sup 3^/µL) in 35% to 72% of cases.5 Basophilia in the blood smear is suggestive of CML rather than ET.86 Teardropshaped erythrocytes and leukoerythroblastosis are suggestive of CIMF or end-stage PV. The red blood cells are usually normochromic and normocytic unless the patient has had significant bleeding.1

Bone marrow biopsy examination is important in the diagnosis of ET. The bone marrow is usually hypercellular but may be normocellular. Stainable iron is present unless the patient has had chronic bleeding. The presence of stainable iron and a normal red blood cell mass is important in ruling out PV. The megakaryocytes in ET are increased in number, enlarged in size, and characteristically have hyperlobated nuclei (Figure 1, A through C). The megakaryocytes also often form small clusters which can be appreciated on the biopsy specimen. Dysplastic and bizarre megakaryocytes are not a usual feature of ET, and the presence of such forms is consistent with CIMF. Megakaryocytic emperipolesis of bone marrow elements may be seen but is not a specific feature. Erythroid or granulocytic hyperplasia is not characteristic, as megakaryocytic differentiation should be the prominent feature. Collagen fibrosis, seen best with a trichrome stain, is absent. Reticulin fibrosis is absent to minimal. Increased neutrophil granulopoiesis, erythroid hyperplasia, significant reticulin fibrosis, or any collagen fibrosis speaks against the diagnosis of ET and suggests the diagnosis may be early PV or CIMF in the cellular or prefibrotic phase.1,7,20,84,87

DIFFERENTIAL DIAGNOSIS

The numerous reactive, hereditary, and neoplastic causes of thrombocytosis must be excluded before making a diagnosis of ET (Figure 2; Table 2). Common neoplastic conditions that can be confused with ET include myelodysplastic syndromes (particularly 5q- syndrome), PV, CML, and CIMF. Reactive thrombocytosis may be secondary to iron deficiency anemia, hemolytic anemia, acute blood loss, underlying or occult malignancy, rebound after withdrawal from alcohol abuse, postsplenectomy, acute or chronic infections, various inflammatory disorders, or drug reactions (vincristine, epinephrine, all-trans-retinoic acid, cytokines, and growth factors), as well as in response to exercise. With iron deficiency, the platelet count rarely exceeds 700 × 10^sup 3^/µL. Chronic inflammatory and infectious disorders that are commonly associated with an elevated platelet count include inflammatory bowel disease, connective tissue disorders, temporal arteritis, tuberculosis, and chronic pneumonitis. Features that favor neoplastic over reactive thrombocytosis include thrombotic events, splenomegaly, no increase in acute phase reactants, megakaryocyte clustering and hyperlobated megakaryocytes in the bone marrow, and abnormal cytogenetic findings, including the detection of JAK2 mutation. It is necessary to demonstrate negative findings such as lack of bone marrow fibrosis, lack of cytogenetic findings seen in MDS or CML, and lack of reactive causes for thrombocytosis.1,7,20,24,27-89

While not part of the true CMPDs, rare families with hereditary thrombocythemia have been identified and studied. These thrombocythemias are inherited in an autosomal dominant fashion as a consequence of germ-line mutations in genes that regulate platelet and megakaryocyte production, and so by definition the cases are due to a nonneoplastic, polyclonal thrombopoiesis. The patients have elevated platelet counts at an early age or at birth with normal neutrophil count and hematocrit. In some patients, mutations in the TPO gene or in the TPO receptor (MPL) gene have been found to be the disease-causing abnormality.90-93 To date, these mutations have not been found in ET. In many patients with hereditary thrombocythemia, the genetic mutation has not been identified.2,94

CONCLUSION

Currently, a definitive diagnosis of ET remains dependent on exclusion of the numerous reactive, hereditary, and neoplastic causes of thrombocytosis. On the other hand, a diagnosis of ET cannot be made in the absence of at least 1 positive diagnostic criterion. Compared to the previous Polycythemia Vera Study Group criteria, the new WHO criteria place more emphasis on the bone marrow biopsy showing increased numbers of enlarged and hyperlobated megakaryocytes in small clusters and minimal to absent marrow reticulin fibrosis. Further basic research and clinical studies are needed to find more specific and sensitive positive findings in ET and to identify possible prognostic factors and targets for therapy.

The identification of new molecular markers in CMPD is a promising direction of study. However, the variable and sometimes inconsistent findings regarding JAK2 and clonality studies suggest that ET, as currently defined, encompasses a heterogeneous mixture of entities. It seems likely that a number of mutations involving thrombopoietin, the MPL receptor, or other genes involved in regulation of megakaryocytopoiesis may result in persistent thrombocytosis with features similar to ET. Incorporation of molecular data could result in a classification system analogous to the WHO classification for acute myelogenous leukemia, based in part on cytogenetic findings. In the future, we may find ET reclassified or subclassified on the basis of JAK2 mutational status or overexpression of PVR-1.

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