Definition and characteristics

Essential (or primary) thrombocythemia (ET) is a chronic myeloproliferative, BCR-ABL1 negative neoplasm (MPN). It is characterized by an increased proliferation of the megakaryocytic lineage in the bone marrow as well as by a dysfunction and a progressive proliferation of platelets in peripheral blood. Due to the lack of specific markers, the diagnosis is not always clear, and therefore secondary thrombocytosis should be excluded. Secondary thrombocytosis is mainly caused by inflammation or iron deficiency.

Up to one third of patients are symptom-free at initial diagnosis of ET, as the diagnosis is often established during a routine blood test. The majority of patients remain symptom-free for several years and have an almost normal life expectancy. In the course of the disease, however, complications such as microcirculation disorders in the sense of circulatory disorders in the hands and feet, thromboembolic complications and bleeding may occur. The risk of thrombosis is increased in the presence of ET. Most cases occur in patients over 60 years of age and is one of the leading causes of death in ET (Elliott et al. 2005). In a small proportion of patients, the disease progresses to polycythaemia vera (PV), post-ET myelofibrosis or myelodysplastic syndrome (MDS) or secondary acute leukemia.

The annual incidence of ET has not been established with certainty and is based on the estimate of the Polycythaemia Vera Study Group (PVSG) for the European region and the USA: 0.2 - 2.3 / 100,000 inhabitants. It occurs at the median age of 50-60 years and is slightly more common in women than in men. Nevertheless, almost 20 percent of patients with essential thrombocythemia are 40 years or younger, and in some cases children and young adults may also be affected (Swerdlow et al. 2017).

Classification

According to the WHO classification 2017, ET belongs to the so-called BCR-ABL1 negative myeloproliferative neoplasms. The clinical differentiation within the MPN is based on the detection of clonal thrombocytosis (see also criteria for essential thrombocytosis). In the peripheral blood smear, platelets are morphologically altered in 90% of patients: enlarged and/or different sizes. A subdivision into subgroups was not made within the WHO classification. However, it should be noted that there are currently no specific disease markers, molecular or otherwise, that uniquely diagnose ET, so a diagnosis should always be based on a combination of clinical, molecular genetic and bone marrow histological findings.

ET WHO Classification 2017

Diagnostic criteria for essential thrombocythemia

Major criteria

  • Platelet count ≥ 450x109/L
  • Bone marrow histology: 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. Very rarely a minor (grade 0-1) increase in reticulin fibres
  • WHO criteria for BCR-ABL1-positive CML, PV, PMF or other myeloid neoplasms are not met
  • JAK2, CALR, or MPL mutation

Minor criteria

  • Presence of clonal marker or
  • Absence of evidence of reactive thrombocytosis

The diagnosis ET requires all four major or the first three major and one minor criterion.

Diagnostics

Cytomorphology

Cytomorphological assessment in MPN involves cellularity in the total and in the individual hematopoietic lineages. It is also important to determine the proportion of blasts. Characteristic changes in ET include

  • Proliferation of megakaryocytes
  • frequently enlarged mature megakaryocytes with hypersegmented (staghorn-like) nuclei without cluster formation
  • predominantly no proliferation of erythrocytes and granulocytes
  • hardly any dysplasia
  • hardly any blasts (< 5%)
  • often a normal cellularity
Chromosome analysis

Chromosomal abnormalities are observed in about 5-10% of patients with ET (Panani et al. 2006, Gangat et al. 2009). These occur rather rarely compared to other MPN. Several studies showed the following recurrent abnormalities in smaller patient cohorts: Trisomy 8 (+8), abnormalities concerning chromosome 9 (+9/del(9q)), deletions in the long arm of chromosome 20 (del(20q)), abnormalities of chromosome 1 and deletions in the long arm of chromosome 5 (del(5q)) (Panani 2006, Gangat et al. 2009, Sever et al. 2009, Swerdlow et al. 2017). Since the presence of a clonal marker is one of the secondary criteria for the diagnosis of an ET according to the WHO classification 2017, the detection of a clonal chromosomal abnormality may be helpful in confirming the diagnosis of an ET. However, due to the small number of cases in the studies, it is difficult to diagnose ET or make a prognostic statement based on cytogenetic factors alone. However, one study showed that the life expectancy of patients who showed chromosomal abnormalities at the initial diagnosis did not differ from those who acquired aberrations over time (Sever et al. 2009). Interestingly, isolated deletions in the long arm of chromosome 5 were observed in MPN, especially in PMF and somewhat less frequently in ET, which are predominantly described in MDS patients (Swerdlow et al. 2017).

FISH

FISH is mainly used as a supplement to cytogenetics in order to be able to make a quantitative statement about the clone size and to obtain a marker suitable for the course of the disease. However, this method can only be used specifically for certain questions, such as the exclusion of a BCR-ABL1 rearrangement within the MPN, and will therefore not be able to replace classical chromosome analysis. Detection of the cytogenetic changes typical for ET can be performed on both blood and bone marrow smears.

Molecular genetics

Mutations in essential thrombocythemia

Table 1: Frequency of different mutations in essential thrombocythemia (Tefferi et al. 2018) 

Mutation

Frequency (%)

JAK2 V617F

55

MPL

3

CALR

20

TET2

5

IDH1/2

1

ASXL1

3

CBL

rare

TP53

2 respectively 27 (in blast crisis)

The exact pathogenesis of ET is not yet fully understood, but as with other MPN mutations in certain genes of a hematopoietic stem cell seem to play a role. In about 50% of patients with ET, a mutation in exon 14 of the Janus kinase-2 (JAK2) gene (JAK2 V617F), which is a member of the tyrosine kinase family and is involved in the signal transduction of erythropoietin, thrombopoietin, and G-CSF, among other things, can be detected (see Table 1). This mutation leads to a permanent activation of JAK2-kinase, which is associated with excessive cell formation. Although the JAK2 mutation is found in half of all ET patients, it is not specific for ET and is also found in other MPN such as PV and in a small percentage (< 5%) of MDS and AML patients (Swerdlow et al. 2017). Therefore, the detection of this mutation is not suitable for the differential diagnosis of individual subtypes within chronic myeloproliferative diseases.

Other mutations, e.g. in the calreticulin (CALR) gene or MPL mutations, which are frequently diagnosed in other MPNs such as primary myelofibrosis (PMF), also occur in ET and significantly more frequently in comparison to PV. JAK2, CALR and MPL are called "driver mutations" and are primarily determined in the diagnostic routine program. If these are negative, further "non-driver" mutations can be examined (see Table 1) and may be helpful in securing the diagnosis of an ET, since the presence of a clonal marker is one of the secondary criteria for the diagnosis of an ET according to the WHO 2017 classification.

Prognosis and risk stratification in essential thrombocythemia

Within chronic myeloproliferative diseases, ET has the most favorable course. Affected patients usually have a normal life expectancy and, especially in the first 10 years, show no difference in quality of life compared to a healthy control population. However, after this decade, a more frequent occurrence of thrombotic events is observed (Wolanskyj et al. 2006). ET should be specifically distinguished from prefibrotic PMF (pre PMF), which was introduced by the WHO in 2001 as a separate class within the MPN. According to the criteria of the Polycythaemia Vera Study Group, some of these pre PMF patients would be classified as ET patients, but have a slightly less favorable course compared to ET (Thiele et al. 2003). Since the probability of fibrotic or leukemic transformation is very small (< 1%, Malecki et al. 2016), the prognostic course of ET is mainly determined by the occurrence of thrombosis and severe bleeding (incidence: 11-39%, Pósfai et al. 2016). Accordingly, most prognostic systems rely on the identification of specific risk factors that can be used to detect patients at risk of thrombosis and bleeding at an early stage. Initially known risk factors include

  • History of known thromboembolic complications or severe bleeding
  • Age over 60 years (it is discussed to raise the age to 65 years, i.e. to take more account of the biological age)
  • Platelet count higher than 1.500.000/µl

Within the ET, two risk groups were initially established and later three and four risk groups respectively. Initially, the above mentioned parameters age and thromboembolic history were the most relevant prognostically.

In subsequent studies, further thrombotic risk factors were investigated in addition to the conventional factors (age and history of thrombosis). These include cardiovascular risk factors (e.g. arterial hypertension, diabetes, obesity and nicotine abuse), leukocytosis and the presence of a JAK2 V617F mutation and its allelic load. Barbui et al. have created an international prognosis system for thrombotic risk factors using a multi-variant analysis in 2012, which was published under the name IPSET (see Table 2). Low risk patients had a thrombosis-free survival of 87% after 15 years, while the high risk group showed a 50% probability of thrombosis-free survival in the first 7 years after diagnosis. The medium risk patients showed similar values to the low risk group during the first 10 years and approached the high risk curve in the following 5 years.

Table 2: ‘International Prognostic Score of thrombosis in essential thrombocythemia’ (IPSET-thrombosis) (Barbui et al. 2012)

Risk factor

HR

Score

Age > 60 years

1,50

1

Cardiovascular risk factors

1,56

1

Previous thrombosis

1,93

2

JAK2 V617F

2,04

2

Low risk

0 - 1

Intermediate risk

2

High risk

≥ 3

The advantage of this new system was that patients could be classified even more precisely and specifically. For example, patients who had been assigned to the high-risk class using conventional parameters (age and symptoms) could be reclassified into the intermediate or even the low risk group. For example: age > 60 years, but no history of thromboembolic events, no cardiovascular risk factors and no JAK2 V617F mutation, would have been high risk in the past, but is now assigned to the low risk class. The IPSET score was validated in a later study in 2016 in 585 ET patients and was refined to include another risk group (see Table 3, Haider et al. 2016). In 2018, the IPSET was applied to a pre-PMF cohort and successfully evaluated to assess the risk of thrombosis in pre-PMF patients (Guglielmelli et al. 2018).

Table 3: Validation of the revised 'International Prognostic Score of thrombosis in essential thrombocythemia (IPSET-thrombosis)' (Haider et al. 2016)

Risk factors

Risk

Age ≤ 60 years

No history of thrombosis

JAK2-wild type

Very low

Age ≤ 60 years

No history of thrombosis

JAK2-Mutation

Low

Age > 60 years

No history of thrombosis

JAK2- wild type

Intermediate

History of thrombosis or

Age > 60 years and JAK2-Mutation

High

Cytogenetic prognostic factors in Polycythaemia vera

Recent studies show that patients with an aberrant karyotype have a significantly worse outcome than patients with a normal karyotype. At the same time, it was shown that during an increasing stage of the disease, i.e. transformation into secondary myelofibrosis or into an accelerated phase or blast crisis, complex aberrant karyotypes were increasingly observed (Tang et al. 2017, Tefferi et al. 2013). Based on these more recent findings, a cytogenetic examination was also increasingly recommended in specialist circles and guidelines at the initial diagnosis and during the course of the disease (Swerdlow et al. 2017).

Table 4: Cytogenetic abnormalities detected at the diagnosis (first bone marrow evaluation) (Tang et al. 2017)

 

Polycythemic phase (n=271)

Post-PV MF (n=112)

AB/BP phase (n=39)

Total (n=422)

Normal karyotype

217 (80%)

62 (55%)

4 (10%)

283 (67%)

Abnormal karyotype

54 (20%)

50 (45%)

35 (90%)

139 (33%)

Single abnormalities

41 (76%)

29 (58%)

5 (14%)

75 (54%)

del(20q)

18

12

1

31

+9

10

0

0

10

+8

6

1

1

8

other (single)

7

16

3

26

Double abnormalities

9 (17%)

9 (18%)

6 (17%)

24 (17%)

+1q

4

7

4

15

other (two)

5

2

2

9

Complex

4 (7%)

12 (24%)

24 (69%)

40 (29%)

del(5q)/-5

0

4

14

18

del(7q)/-7

1

2

15

18

del(17p)/-17/i(17q)

1

4

9

14

AP/BP: accelerated/blast phase; Post-PV MF: post-polycythemic myelofibrois.

Molecular Genetic Prognostic Factors in Essential Thrombocythemia

Because the prognosis of ET patients is highly dependent on the risk of thrombosis, no major scoring system - such as the MIPSS70+ for PMF - involving clinical, cytogenetic and molecular genetic prognostic factors has yet been introduced (Tefferi et al. 2108). JAK2 mutation can be detected in about 50% of ET patients, which is also frequently observed in all other MPNs. Due to the frequency of the JAK2 mutation, the prognostic relevance of the JAK2 mutation was investigated. However, studies have shown controversial results: an association between an existing JAK2 mutation and a higher Hb and leukocyte count and thus a significantly higher risk of thrombosis has been described (Campbell et al. 2005, Finazzi et al. 2006). Further studies, however, showed that the JAK2 mutation does not appear to be associated with the incidence of thromboembolic events (Antonioli et al. 2005, Carobbio et al. 2007). These differences may be due to: insufficient clinical criteria to adequately differentiate ET and PV patients, different patient selection and study design, and sole focus on qualitative JAK2 V617F expression rather than quantitative allelic load. Therefore, in subsequent studies, the allelic load was investigated. 2-4% of patients with ET have a homozygous JAK2 mutation. It was shown that patients with a homozygous mutation had a significantly higher risk to get thromboembolic events compared to heterozygous mutated or wild type patients. The higher the allelic load, the higher the risk of thrombosis (Kittur et al. 2007, Vannucchi et al. 2007, Antonioli et al. 2008). In addition to the JAK2 mutation, a CALR mutation is detected in approximately 20% of all ET patients. Calreticulin is a chaperone protected protein involved in differentiation, apoptosis and cell division. Compared to the JAK2 mutation, patients with a CALR mutation showed a lower risk of thrombosis (Torregrosa et al. 2016). However, MPL mutations occur in only 5% of patients, and no precise data on the risk of thrombosis in this patient group are known. The number of patients in whom none of the three "driver mutations" could be detected ("triple-negative") is estimated at 10-25%. These patients also showed an increased risk of thrombosis (Ju et al. 2018).

In addition to the above-mentioned "driver mutations", further studies investigated "non-driver mutations" during the course of the disease and their influence on overall survival as well as the probability of transformation into secondary myelofibrosis or acute leukemia. The most frequent mutations besides the already mentioned "driver mutations" JAK2/CALR/MPL were mutations in the genes ASXL1 (20%), TET2 (11%), DNMT3A (7%) and SF3B1 (5%). For other genes the following frequencies were found: SRSF2: 2%, EZH2: 2%, U2AF1: 1%, RUNX1: 1% and TP53: 2% (Tefferi et al. 2019). Table 5 lists the mutations examined by Tefferi et al. that showed an unfavorable influence on overall survival (OS), leukemia-free survival (LFS) and myelofibrosis-free survival (MFFS).

Table 4: "Non-driver mutations" and their prognostic significance (Tefferi et al. 2019)

 

Mutations

Adverse effect on OS

SF3B1, SRSF2, EZH2, U2AF1

Adverse effect on LFS

SRSF2, EZH2, TP53, RUNX1

Adverse effect on MFFS

SF3B1, U2AF1

OS: Overall survival, LFS: Leukemia-free survival, MFFS: Myelofibrosis-free survival.

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