HAEM4:Essential Thrombocythemia (ET)

Primary Author(s)*

Rebecca Smith, PhD, Director, Genetics Associates, Nashville, TN

Cancer Category/Type

Myeloproliferative Neoplasm

Cancer Sub-Classification / Subtype

Essential Thrombocythemia (ET)

Definition / Description of Disease

Essential Thromocythemia (ET) is one of the BCR-ABL negative myeloproliferative neoplasm (MPN) that is characterized by clonal expansion of differentiated myeloid cells driven by a somatic mutations. ET is characterized by an increased number of platelets in the blood (Platelet count >= 450 x10⁹/L). ET is associated with proliferation of platelet precursors (large, mature, megakaryocytes) in the bone marrow. Because of this increase in platelets complications frequently include blood clotting and/ or bleeding^[1]. The most common disease consideration of ET is an increased risk of vascular complications^[2]

A diagnosis of Essential Thromocythemia (ET) requires meeting all 4 major criteria or the first 3 major and the minor criteria^[1]^[3] :

Major criteria
· Platelet count of >450 x 10⁹ /L
· Bone Marrow biopsy showing proliferation mainly of the megakaryocytic lineage with increased numbers of enlarged, mature megakaryocytes with hyperlobulated nuclei; no significant increase or left shift in neutrophil granulopoiesis or erythropoiesis; very rarely a minor (grade 1) increase in reticulin fibers
· WHO criteria for BCR-ABL-positive chronic myeloid leukemia, polycythemia vera, primary myelofibrosis, or other myeloid neoplasms are not met.
· JAK2, CALR, or MPL mutation
Minor criteria
· Presence of a clonal marker or absence of evidence of reactive thrombocytosis

Synonyms / Terminology

Idiopathic thromcythemia/ thrombocytosis

Essential haemorrhagic thrombocythemia

Idiopathic haemorpagic thrombocythemia

Idiopathic thrombocythemia

Essential thrombocytosis

Epidemiology / Prevalence

Essential Thrombocythemia is the most common type of Myeloproliferative Neoplasm. The estimated ET annual rate of developing the condition in the United States is 2.5 cases per 100,000. The prevalence is estimated to be 24 cases per 100,000 population annually^[2]^[4]. The disease occurrence increases with age, with most patients presenting between ages 50-60 years old. The incidence is higher in women then in men with an approximate 2:1 ratio^[2].

Clinical Features

Approximately half of ET cases are asymptomatic at the time of diagnosis and are identified by an elevated platelet count by a routine peripheral blood count. The other half is diagnosed after a vascular occlusion or hemorrhage. Other cases present with symptoms including transient ischaemic attacks, and thrombosis of major arteries and veins. Mild splenomegaly is present in approximately 50% of cases at diagnosis and hepatomegaly in 15-20 %^[1].

ET is characterized by sustained high platelet counts, and the current threshold (≥ 450 x10⁹/L) exceeds the 95^th percentile for normal platelet counts. Because this cut-off is at the upper range of normal, it is important to meet all the diagnostic criteria in order to rule out other neoplastic and non-neoplastic causes of thrombocytosis.

In a recent study, the most frequent ET patient symptoms were reported to be fatigue (90.3%), numbness (58.8%), insomnia (58%), sad mood (57.3%), vertigo (56,1%), concentration problem (55.8%), inactivity (53.7%), early satiety (53.2%), night sweats (51.3%), sexual problem (51.0%), headache (47.1% ), abdominal discomfort (45.3%), bone pain (45.2%), cough (41.4%), itching (40.6%), abdominal pain (38.2%), weight loss (23.4%) and fever (17%)^[2].

Sites of Involvement

The cell of origin is proposed to the hematopoietic stem cell and megakarytocytes can be seen in the bone marrow. The most dominant feature is marked thrombocytosis in the peripheral blood.^[1]

Morphologic Features

In ET, the elevated platelets display different sizes, ranging in size from tiny to gigantic. The white blood cell count and red blood cells are usually normal. Within the bone marrow of ET patients, a marked proliferation of megakaryocytes is seen. These megakaryocytes are predominantly large to giant in size and show abundant, mature cytoplasm and deeply lobed and hypersegmented (staghorn-like) nuclei. These megakaryocytes are typically dispersed throughout the bone marrow, but may occur in loose clusters. Bone marrow aspirate smears from ET patients also reveal markedly increased numbers of large megakaryocytes with hyperlobulated nuclei, as well as large sheets of platelets in the background. Although not a specifici findhing, emperipolesis, or the presence of an intact cell within the cytoplasm of another cell, is frequently observed in ET^[1].

Properly diagnosing ET is critical for proper treatment and prognosis. Morphology of the peripheral blood smear and bone marrow can be used to differentiate ET between other diseases^[1]:

Large dense clusters are very rarely found in ET but occur in pre-PMF and overly in primary myelofibrosis
No increase in myeloblasts and no myelodysplasia
The network of reticulin fibers is usually normal (rarely increased, but never more than WHO grade 1)
Combined granulocytic and erythroid proliferation could suggest polycythemia vera
Significant dyserythropoiesis and dysgranulopoiesis suggests a diagnosis of myelodysplastic syndrome rather than ET
The large megakaryocytes of ET can be distinguished from other pathologies:
- Medium-sized non-lobated megakaryocytes seen in myelodysplastic syndrome with isolated del(5q)
- Small dysplastic megakaryotcytes seen in AML or MDS with inv(3)(q21q26.2) or t(3;3)(q21;q26.2)
- Small (dwarf) megakaryocytes of chronic myeloid leukemia

Immunophenotype

Flow cytometery results of ET samples typically includes normal number of phenotype myeloblasts with normal myeloid scatter by CD45/ SSC. There is no immunophenotypic evidence of myelodysplasia, (normal CD10/ CD13/ CD16/ CD11b myeloid maturation pattern). There is no evidence for monoclonal B cell lymphoproliferative disease and no unusual T cell phenotypes are identified^[5].

Chromosomal Rearrangements (Gene Fusions)

Very rarely, cases of ET acquire a BCR-ABL1 rearrangement. Consequently, in this situation, a morphological and hematological shift capable of chronic myeloid leukemia evolution may occur^[6]

Characteristic Chromosomal Aberrations / Patterns

An abnormal karyotype is found in 5-10% of cases; however, there is no consistent abnormality. Reported, but non-specific, abnormalities in ET include gain of chromosome 8, abnormalities of 9q, and del(20q). Isolated instances of del(5q) have also been reported in ET^[1].

Genomic Gain/Loss/LOH

Uniparental disomy (UPD) in Essential Thrombocythemia has not been reported. Conversely, UPD of chromosome 9p (typically causing homozygosity of JAK2 pathogenic variants) is a frequent clonal occurrence in polycythemia vera (PV) ^[7].

Gene Mutations (SNV/INDEL)

Most cases of ET harbor a phenotypic driver mutation in JAK2 (50-60% of ET cases), CALR (in 30%), or MPL (in 3%), and about 13% of ET are triple negative for these mutations. None of these mutations are specific for ET; however, the presence of these mutations would rule out reactive or secondary thrombocytosis^[1].

Gene	Mutation	Percent of ET cases	Oncogene/Tumor Suppressor/Other	Presumed Mechanism (LOF/GOF/Other; Driver/Passenger)
JAK2	V617F, c.1849G>T	50-60%	Non-receptor tyrosine kinase	Constitutively activated
CALR exon 9	Insertion or deletion (52-bp insertion, or 5 bp deletion) are most prevalent	30%	Typical function as a ER CA2+ binding chaperone. Pathogenic variants cause ectopic cellular function in hematopoietic pathway.	GOF
MPL exon 10	Most common affect a tryptophan in position 515 (W515L, W515K, W515A, and W515R).	3%	proto-oncogene receptor	Constitutively activated
Triple negative	No specific marker (negative for pathogenic variants in JAK2, CALR, and MPL)	12%	Not available	Not available

Other Mutations

These main driver mutations, JAK2, CALR, and MPL have been considered to be mutually exclusive; however, there is a recent report of additional mutations in CALR or MPL co-existing in approximately 10% of patients with low JAK2 burden. The clinical significance of the co-occurrence of these variants is still unclear^[8].

Sub-clonal mutations have been seen in TET2, ASXL1, CBL, IDH and IKZF1 are observed however the diagnostic value of these mutations in ET are not clear. In PMF, however, these mutations have a negative prognostic value and their presence is associated with lower survival and a greater risk of acute leukemia evolution (Accurso et al. 2019, Kalmpfl et al. 2013)

Type	Gene/Region/Other	Comments
Secondary Mutations	TET2, ASXL1, CBL, IDH and IKZF1, DNMT3A, SF3B1, CEBPA, TP53, SH2B3, EZH2, and CSF3R.	The impact and disease prognosis of these secondary mutations in ET is not clear. These variants are not specific and overlap in occurrence with other MPNs, MDS, and AML diseases.
Mutually Exclusive	These main driver mutations, JAK2, CALR, and MPL have been considered to be mutually exclusive; however, a recent reports of additional mutations in CALR or MPL co-existing in approximately 10% of patients with low JAK2 burden.	The clinical significance of co-occurring variants is unclear^[8]

Epigenomics (Methylation)

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Genes and Main Pathways Involved

JAK2

Janus Kinase 2 (JAK2) is a non-receptor kinase that activates the JAK-STAT pathway upon ligand binding. JAK2 has a critical role in signaling for hematopoietic cytokinases and associates to the cytoplasmic region of multiple receptors involved in myelopoesis including erythropoietin (Epo), thrombopoietin, granulocyte colony stimulating factor, IL-3, and granulocyte macrophage colony stimulating factor receptors). After ligand binding to these receptors, JAK2 tyrosine kinase domain is activated and allows for progression of the signal through signal transducers and activators of transcriptions (STATs), Ras-mitogen-activated protein kinase (MAPK), and phosphoinositide-S3-kinasae pathways, which regulate proliferation, apoptosis, and differentiation in myeloid cells^[9].

The most prominent pathogenic variant in ET, JAK2 V617F, makes up 50-60% of cases and occurs at nucleotide 1849 in exon 14, and is a G to T somatic transversion. This pathogenic variant occurs in the pseudokinase JH2 domain of the protein, inhibits the normal JH2 autoinhibitory mechanism, and consequently results in the constitutive activity of JAK2^[10]. The allelic burden of JAK2 at diagnosis can provide prognostic information because it correlates with phenotypic severity of MPNs, the risk of thrombotic events, and progress to secondary myelofibrosis (MF)^[9].

CALR

Calreticulin (CALR) pathogenic variants represent the second most common genetic abnormality in ET and is found in ~30% of cases. CALR is a Ca2+ binding protein with chaperone activity that resides predominantly in the endoplasmic reticulum. In its wildtype form, CALR has a variety of roles including protein quality control, calcium metabolism, immune response, phagocytosis, cell adhesion and others. The localization of the CALR protein is determined by a KDEL sequence on the C-terminal end of the protein^[9].

Pathogenic CALR variants driving MPNs, including more than 50 different variants, typically consisting of an insertion or deletion in exon 9 causing a frameshift. This results in a novel C-terminal peptide sequence, loss of the KDEL signal, and partial dislocation from the endoplasmic reticulum. There are two predominate variants seen driving ET (and MPNs as a whole), a type 1 variant (L367fs*46) results from a 52-bp deletion and a type 2 variant (K385fs*47) that results from a 5-bp TTGTC insertion. Within ET patients, the distribution of is 57% of patients have a type 1 variant (or type 1-like) and 39% were found to have a type 2 variant (or type 2-like)^[9].

MPL

The myeloproliferative leukemia virus oncogene (MPL) encodes for the receptor for the thrombopoietin (TPO) ligand. This receptor is a primary regulator of megakaryopoiesis, driving early stages of megakarytocyte differentiation, and hematopoietic stem cell survival and proliferation. TPO binding to MPL leads to the activation of JAK2, leading to the downstream activation of signaling pathways involving STAT 1, 3, and 5, Akt, and ERK 1/2^[9]^[11].

Mutations in MPL have been reported in 1-4% of patients with ET. Constitutively activating mutations in MPL all cluster in exon 10 and the majority of the cases affect a tryptophan in position 515 (W515L, W515K, W515A, and W515R)^[9]. In a recent study, MPL-mutated ET was reported to have a higher rate of fibrotic progression than patients with a MPL-wildtype ET disease^[12].

Driver mutations: CALR vs JAK2

Essential Thrombocythemia (ET) with CALR pathogenic variants has been proposed to be a distinct entity with a less severe course of disease compared with JAK2 mutations. ET patients with CALR-pathogenic variants were more often found to exhibit lower leukocyte and hemoglobin values, higher platelet counts, and to be male compared with JAK2-mutated ET patients. Additionally, a lower incidence of thrombosis was seen in CALR-mutated ET vs. JAK2-mutated ET. This information has the potential to further stratify ET patients based on the prognosis and severity of the disease ^[13]^[14].

Diagnostic Testing Methods

Samples from patients diagnosed with ET are routinely tested for JAK2 V617F, CALR exon 9 insertions or deletions, and MPL exon 10 gain of function variants. Although not an exhaustive list of testing methods for each pathogenic variant, several detection strategies have been described (see below)^[9]:

Genetic change:	Methods for testing
JAK2 V617F	Allele specific assay, TaqMan assay, NGS, real-time allele specific PCR,
CALR exon 9 insertion or deletion	Sanger sequencing Fragment length analysis, NGS, Real-time qPCR, HRM, ddPCR, pyrosequencing
MPL exon 10	Sanger sequencing, pyrosequencing, melt-curve analysis, NGS, allele specific qPCR, a multiplexes allele-specific PCR assay for the four most frequent MPL exon 10 mutations (W515L, W515K, W515A, and S505N)

Clinical Significance (Diagnosis, Prognosis and Therapeutic Implications)

ET is characterized by overall favorable prognosis as compared to the other MPNs (PV and PMF) with an expected survival of 19.8 years; however, life-expectancy is still reduced compared to the control population. Incidence of blast transformation and fibrotic transformation is lower in ET as compared to PV^[2]. Transformation of ET to a blast phase (AML or MDS) occurs in <5% of cases, and is likely related to previous cytotoxic therapy^[1].

Treatment for ET is usually kept to a minimum and the patient is monitored for progression to leukemia and post-ET myelofibrosis. The main indication for treatment of ET is to prevent thrombosis. There are four major risk categories for thrombosis risk^[15]:

High-risk: presence of thrombosis history or presence of both JAK2/MPL mutations and advanced age
Intermediate-risk: presence of advanced age
Low risk: presence of JAK2/MPL mutations
Very low risk: absence of all risk factors

Disease progression

The transformation of ET to AML and/or post-ET myelofibrosis (post-ET MF) are rare events. The risk of transformation to acute leukemia was reported to 2-3% at 10 years and 5% at 15 years after diagnosis. Risk factors included age and age over 60 years. Occurrence of post-ET myelofibrosis does occur rarely with a risk of 6% at 15 years. Anemia was identified as the only significant risk factor for development of post-ET myelofibrosis^[16]

Familial Forms

Most cases of ET are sporadic however, familial clustering has also been observed. One study established a 6x relative risk of developing a MPN, when there is also an affected relative^[17] The JAK2 GGCC 46/1 haplotype represents a low penetrance predisposition allele and is associated with JAK2 mutated MPN. This haplotype consists of analyzing four SNPs in linkage disequilibrium, and located in introns 10, 12, 15 and 15 of the JAK2 gene and is found in about 50% of healthy subjects ^[18].

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