Polycythaemia vera (PV)

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Cytomorphology
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Immunophenotyping
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Chromosome analysis
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FISH
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Molecular genetics
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Characteristics
Classification
Diagnostics
Prognosis
Recommendation

Polycythaemia vera (PV) is a myeloproliferative, BCR-ABL1-negative neoplasms. Although hyperplasia of all three cell series (leukocytes, erythrocytes and thrombocytes; so-called panmyelosis) is usually present, Polycythaemia vera is characterized by a dominant proliferation of erythrocytes, which is why secondary erythrocytosis should always be excluded by differential diagnosis. Secondary erythrocytosis primarily caused by stress, smoking, cardiac causes and infections. The Polycythaemia vera variant with exclusive proliferation of the red cell series is called polycythaemia vera rubra, but occurs only very rarely. Furthermore, a "masked" form of Polycythaemia vera has been described, which has lower haemoglobin and haematocrit values than the form originally defined in the WHO 2008 (Barbui et al. 2014).

Characteristics of PV

Polycythaemia vera is clinically divided into two phases: A chronic phase (prepolycythemic and polycythemic phase), which is characterized by an overproduction of erythrocytes and associated elevated haemoglobin and haematocrit values. This is followed by the so-called late phase, in which the disease progresses into secondary myelofibrosis (post-PV myelofibrosis). After a median observation period of 10 years, the rate of post-PV MF is about 15%, after 20 years it is 50%. This is accompanied by cytopenia, extramedullary haematopoiesis in the spleen, liver and other organs, and consecutive splenomegaly. In a small proportion of patients (4%), a transition to an acceleration, also known as the blast phase (blast percentage 10-19%) or a blast crisis (blast percentage >20%) may occur (Alvarez-Larrán et al. 2009, Passamonti et al. 2010, Tefferi et al. 2013).

The annual incidence of PV is 0.01-2.8 / 100,000 inhabitants in Europe and North America and occurs predominantly in the median age range of 60-65 years (Swerdlow et al. 2017).

Classification of Polycythaemia vera

According to the WHO classification 2017, Polycythaemia vera belongs to the so-called BCR-ABL1 negative neoplasms. The clinical differentiation within myeloproliferative neoplasms is based on the detection of clonal erythrocytosis (see also criteria for the diagnosis of polycythaemia vera). A division into subgroups was not made within the WHO classification. In the current version of the WHO, the haemoglobin and haematocrit limits have been adjusted in order to also be able to diagnose cases with a so-called "masked" form at an early stage. However, it should be noted that there are currently no specific disease markers, molecular or otherwise, that can uniquely diagnose Polycythaemia vera, so a diagnosis should always be based on a combination of clinical and bone marrow histological findings.

Polycythaemia Vera WHO Classification 2017

Myeloproliferative Neoplasms (MPN)

Polycythaemia vera (PV):

Chronic phase (pre-polycythaemic and polycythaemic phase)
Late phase (post-PV myelofibrosis)

Diagnostic criteria for Polycythaemia vera

Major criteria

Elevated haemoglobin concentration (♂: >16.5 g/dL, ♀: >16g/dL) or elevated haematocrit (♂: >49%, ♀: >48%),*
Bone marrow biopse showing trilinear myeloproliferation with pleomorphic megakaryopoiesis
Presence of JAK2 gene (V617F or exon 12) mutation

Minor criteria

Subnormal serum erythropoietin level

The Polycythaemia vera diagnosis requires either all three main criteria or the first two main criteria and the secondary criterion.

* In cases with persistent erythrocytosis (♂: haemoglobin >18.5 g/dL or haematocrit >55.5%, ♀: haemoglobin >16.5 g/dL or haematocrit >49.5%), a bone marrow biopsy may not be required if a JAK2 mutation (major criterion 3) has been detected and the erythropoietin level (minor criterion) is reduced.

** The determination of erythrocyte mass with ⁵¹Cr-labelled erythrocytes allows the distinction between true polyglobulia and pseudopolyglobulia. This method is not routine in Germany. The gender-specific threshold values for haemoglobin defined in the WHO criteria have only established themselves to a limited extent in Germany. Relatively widespread use is made of an elevated haematocrit in both men and women.

95%

of patients with Polycythaemia vera have a JAK2 V617F mutation and round about 3% have a JAK2 Exon12 mutation
(Onkopedia guideline PMF).

Polycythaemia vera - Diagnostics

The cytomorphological assessment in MPN involves cellularity in the total as well as in the individual haematopoietic series. It is also important to determine the blasts in order to differentiate between acceleration (blasts 10 - 19%) or blast crisis (blasts >20%).

Among the characteristic changes in Polycythaemia vera are:

Clear hypercellularity
Granulopoiesis increased in quantity, left-shifted
Erythropoiesis increased in quantity, left-shifted
Megakaryopoiesis increased in quantity, cells tend to form loose clusters, frequently hypersegmented nuclei
Reduced iron storage in the BM

Chromosomal aberrations are observed in 14 - 20% of patients with Polycythaemia vera at initial diagnosis (Sever et al. 2013, Gangat et al. 2008, Swolin et al. 1988) and in about 20% in a chronic phase (Tang et al. 2017). In contrast, cytogenetic changes are observed in 45% and 90% of patients in an advanced or accelerated phase (Tang et al. 2017). At initial diagnosis, deletion in the long arm of chromosome 20 (del(20q)), trisomy 8 (+8) and trisomy 9 (+9) are most frequently observed. Furthermore, deletions in the long arm of chromosome 13 (del(13q)), deletions in the short arm of chromosome 12 (del(12p)) and gains of material of the long arm of chromosome 1 (+1q) have been described (Cerquozzi et al. 2015, Reilly 2008, s et al. 2013, Swerdlow et al. 2017, Swolin et al. 2008). Recent studies, including one of the International Working Group for Myeloproliferative Neoplasms Research and Treatment (IWG-MRI) showed that patients with an aberrant karyotype had a higher risk of progression and a worse overall course (Tang et al. 2017, Tefferi et al. 2013, Dingli et al. 2006). As the disease progressed, increasingly complex karyotypes were observed, which mainly showed chromosomal changes in the long arm of chromosome 5 (del(5q)), aberrations of chromosome 7 (-7/del(7q)), aberrations of chromosome 17 (-17/del(17p)/add(17p)) and monosomy 18 (-18) (Tang et al. 2017). However, none of these aberrations are specific for Polycythaemia vera , they also occur in other MPN and also in MDS and AML (Swerdlow et al. 2017).

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 myeloproliferative neoplasms, and will therefore not be able to replace classical chromosome analysis. Detection of the cytogenetic changes typical for Polycythaemia vera can be performed on both blood and bone marrow smears.

JAK2 V617F mutations in Polycythaemia vera

Table 1: Mutational frequency in Polycythaemia vera (Tefferi et al. (1) 2018)

Gene mutations	Frequency(%)
JAK2 V617F (exon 14)	96
JAK2 exon 12	3
MPL	0
CALR	0
TET2	16
IDH1/2	2
DNMT3A	7
ASXL1	3
EZH2	3
CBL	rare
TP53	27 (in blast crisis)

The exact pathogenesis of Polycythaemia vera has not yet been fully elucidated, but mutations in certain genes of a haematopoietic stem cell and the resulting clonal haematopoiesis seem to play a role. In 96% of patients with Polycythaemia vera, a mutation in exon 14 of the Janus kinase-2 (JAK2) gene, which is a member of the tyrosine kinase family and is involved in the signal transduction for erythropoietin, thrombopoietin, and G-CSF, among other things, has been detected. This mutation leads to a permanent activation of the JAK2 kinase, which is associated with excessive cell formation. If this cannot be detected, a mutation may be present in exon 12 of the JAK2 gene (see Table 1). Although the JAK2 mutation is found in a large percentage of Polycythaemia vera patients, it is not specific for PV and can also be diagnosed in other MPN and in a small percentage (< 5%) of MDS and AML patients (Swerdlow et al. 2017). Other mutations, e.g. in the calreticulin (CALR) gene or MPL mutations, which are frequently diagnosed in other MPNs such as primary myelofibrosis, occur rather rarely in Polycythaemia vera. 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 investigated (see Table 1).

Prognosis and risk stratification of Polycythaemia vera

The prognosis varies greatly among Polycythaemia vera patients, which is why the risk factors that affect survival expectation are largely assessed individually for each patient. Untreated patients with Polycythaemia vera show an extremely shortened life expectancy (1.5 years) compared to treated patients (median survival between 14 and 19 years) (Tefferi et al. 2019). Arterial or venous thromboembolism is the most frequent cause of morbidity and death (40% in treated patients and 60% in untreated patients) followed by the complications of progression in the sense of secondary myelofibrosis or the development of a blast crisis (Gruppo italiano 1995, Chiewitz et al. 1962).

In more recent studies, retrospective analyses have been used to try to establish different prognostic scores for survival, but no really uniform picture has emerged, which is why risk stratification for therapy decisions continues to be based on the risk of thrombosis and prognostic models have developed primarily on the basis of clinical data (Bonicelli et al. 2013, Tefferi et al. 2013, Vannucchi et al. 2018 (see Table 2)). Thus, a distinction is often made between a high and a low risk of thrombosis. Assured risk factors for thromboembolism are: advanced age (≥60 years) and an arterial or venous thrombosis that has already occurred. Assured risk factors for a leukemic transformation are also advanced age (≥60 years), leukocytosis and aberrant karyotype.

Table 2: Risk stratification in patients with Polycythaemia vera

(Vannucchi et al. 2018)

Variable	Risk categories	Endpoints of the score	Use for risk adapted therapy
Age ≥ 60 Thrombosis (at or before diagnosis)	low (neither of these) high (both)risk of thrombosis		Yes
Age (57–66 years = 2 points) (67 ≥ years = 5 points) Leukocytes ≥15 x 10⁹/l (= 1 point) Venous thrombosis (= 1 point)	low (0 points) Intermediate (1–2 points) high (≥ 3 points)	overall survival	No
Leukocytosis JAK2 V617F allele burden Generic CV risk factors			Not yet formally in risk assessments included

CV, cardiovascular; *cardiovascular risk factors: hypertension, diabetes and smoking.

Tefferi et al. have also developed a prognostic three-step model using the parameters leukocyte count, age, venous thrombosis, leukoerythroblastic blood smears, thrombocytosis and itching. Patients with elevated leukocyte count, venous thrombosis and leukoerythroblastic blood smears showed a negative prognosis independent of age, while thrombocytosis and itching were associated with better survival. Within the system, points are also awarded which divide patients into low (0 points), intermediate (1 or 2 points) and high risk (≥ 3 points) groups. These points are awarded as follows: Age ≥67 years (5 points), age 57-66 years (2 points), leukocyte count ≥ 15 x 10⁹/l (1 point) and venous thrombosis (1 point) (Tefferi et al. 2013).

However, since patients are primarily at risk of developing secondary myelofibrosis (SMF), the group around Passamonti developed a "Myelofibrosis Secondary to PV and ET-Prognostic Model (MYSEC-PM)", which classifies patients into a total of four risk categories with regard to overall survival: low (< 11 points), intermediate 1 (11 - 13 points), intermediate 2 (14 - 15 points) and high risk (≥ 16 points). The parameters listed in Table 3 were used for categorization and scoring: Age, haemoglobin values, platelet values, number of circulating blasts, no CALR mutation and constitutional symptoms (fever, weight loss, night sweats, etc.) (Passamonti et al. 2017).

Table 3: Results oft he multivariable analysis to define predictors of inferior survival in 685 annotated patients with post essential thrombocythemia and post polycythemia vera myelofibrosis

(Passamonti et al. 2017)

Covariates	HR	95% CI	P Value	Risk coefficient Beta	Points assigned in the MYSEC-prognostic model
Age at SMF diagnosis	1.07	1.05–1.09	<0.0001	0.068	0.15^a
Hemoglobin <11 g/dl	2.3	1.6–3.3	<0.0001	0.8	2
Platelets <150 × 10⁹/l	1.7	1.2–2.5	0.006	0.5	1
Circulating blast cells ≥3%	2.9	1.8–4.8	<0.0001	1.1	2
**CALR-unmutated genotype**	2.6	1.2–5.3	0.001	0.9	2
Constitutional symptoms	1.5	1.0–2.0	0.03	0.4	1

Abbreviations: CI, confidence interval; HR, Hazard Ratio, SMF: secondary myelofibrosis.
^aAge-related risk points. They are listed (per year) for comparison with the other factors (0.15 points per patient’s year of age).

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%)
Aberrant 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
Andere (einfach)	7	16	3	26
Two abnormalities	9 (17%)	9 (18%)	6 (17%)	24 (17%)
+1q	4	7	4	15
other (two times)	5	2	2	9
Komplex	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 for Polycythaemia vera

To date, no general scoring system, such as MIPSS70+ for primary myelofibrosis, has been introduced for Polycythaemia vera, which includes clinical, cytogenetic and molecular genetic prognostic factors (Tefferi et al. (2) 2018).

In 96% of Polycythaemia vera patients a JAK2 mutation was found, which was a major breakthrough in the diagnosis of Polycythaemia vera, but initially did not show great prognostic relevance. In a more recent study, however, Passamonti et al. were able to show that a JAK2 V617F allelic load of over 50% seems to be associated with fibrotic transformation (Passamonti et al. 2010). In another study by Ortmann et al. it was found that the order in which two different mutations are acquired has a significant influence on the clinical course of the patients. In this case JAK2 V617Fund TET2 mutations were investigated: Patients with an initial JAK2 mutation showed a higher risk of thrombosis, while patients with an initial TET2 mutation presented a more indolent course (Ortmann et al. 2015).

Further studies investigated various mutations, both "driver" and "non-driver" mutations, in the course and influence of these mutations on overall survival and the likelihood of transformation into secondary myelofibrosis or a blast phase or blast crisis. If several mutations are already present in addition to one of the "driver" mutations at the time of initial diagnosis, this increases the risk of a blast phase. Furthermore, it was shown over the course of the study that significantly more mutations are acquired (25.6 mutations x 100 person-years) compared to patients who had a small number of additional mutations at initial diagnosis (1.7 mutations x 100 person-years). In addition, patients with additional mutations at initial diagnosis were more likely to develop cytopenia under hydroxyurea therapy and thus have a higher risk of developing AML. The following "non-driver" mutations were most frequently detected in Polycythaemia vera: TET2, DNMT3A, TP53 and ASXL1. Patients with ASXL1, TP53, SRSF2, IDH1/2 and RUNX1 showed a higher risk of transforming into AML, while mutations in SF3B1 and IDH1/2 and a high JAK2 V617F allelic load were associated with a higher risk of transforming into myelofibrosis (Senin et al. 2017, Tefferi et al. 2016).

Tabelle 5: „Non-Driver“ Mutations and their prognostic significance

(Senin et al. 2017)

	Genes
Higher probability of developing cytopenia	DNMT3A, SRSF2, IDH1/2, RUNX1, TP53
Higher probability of mutation acquisition in the course	SRSF2, IDH1/2, RUNX1 Overall high number of mutations at initial diagnosis
Significantly shorter survival	DNMT3A, SRSF2, SF3B1, IDH1/2, RUNX1
Higher probability of MF transformation (mutation at initial diagnosis)	SF3B1, IDH1/2 High JAK2 V617F allele load
Higher probability of an AML transformation (mutation at initial diagnosis)	ASXL1, TP53, SRSF2, IDH1/2, RUNX1 Overall high number of mutations at initial diagnosis

Polycythaemia vera - Recommendation

Besides the collection of clinical and laboratory chemical parameters, histological and cytomorphological examination of bone marrow and blood, cytogenetic analysis, and molecular genetic examinations (JAK2 V617F mutation, if negative, exon 12 of the JAK2 gene, if negative, CALR and MPL and non-driver mutations should also be examined) are recommended.

Alvarez-Larrán A et al. Postpolycythaemic myelofibrosis: frequency and risk factors for this complication in 116 patients. British Journal of Hematology 2009;146:504-509.

Barbui T et al. Masked polycythemia vera diagnosed according to WHO and BCSH classification. American Journal of Hematology 2014;89:199-202.

Bonicelli G et al. Leucocytosis and thrombosis at diagnosis are associated with poor survival in polycythaemia vera: a population-based study of 327 patients. British Journal of Haematology 2013;160:251-254.

Cerquozzi S et al. Blast transformation and fibrotic progression in polycythemia vera and essential thrombocythemia: a literature review of incidence and risk factors. Blood Cancer Journal 2015;5:e366.

Chiewitz E et al. Complications and causes of death in polycythemia vera. Acta Med Scand 1962,172:513-523.

Diez-Martin JL et al. Chromosome studies in 104 patients with polycythemia vera. Mayo Clinic Proceedings 1991;66(3):287-299.

Dingli D et al. Presence of unfavourable cytogenetic abnormalities is the strongest predictor of poor survival in secondary myelofibrosis. Cancer 2006;106(9):1985-1989.

Gangat N et al. Cytogenetic studies at diagnosis in polycythemia vera: clinical and JAK2V617F allele burden correlates. European journal of Haematology 2008;80(3):197-200.

Gruppo Italiano Studio Polycythemia vera: The natural history of 1213 patients followed for 20 years. Annals of Intern Medicine 1995,123:656-666.

Ortmann CA et al. Effect of mutation order on myeloproliferative neoplasms. New English Journal of Medicine 2015;372:601-612.

Passamonti F et al. A prospective study of 338 patients with polycythemia vera: the impact of JAK2 (V617F) allele burden and leukocytosis on fibrotic or leukemic disease transformation and vascular complications. Leukemia 2010;24:1574-1579.

Passamonti F et al. A clinical-molecular prognostic model to predict survival in patients with post polycythemia vera and post essential thrombocythemia myelofibrosis. Leukemia 2017;31:2726-2731.

Reilly JT. Pathogenetic insight and prognostic information from standard and molecular cytogenetic studies in the BCR-ABL-negative myeloproliferative neoplasms (MPNs). Leukemia 2008;22(10):1818-1827.

Senin A et al. Non-driver mutations in patients with JAK2V617F-mutated polycythemia vera or essential thrombocythemia with long-term molecular follow-up. Annals of Hematology 2018;97;443-451.

Sever M et al. Significance of cytogenetic abnormalities in patients with polycythemia vera. Leukemia and Lymphoma 2013;54(12):2667-2670.

Swerdlow SH et al. WHO classification of tumours of haematopoetic and lymphoid tissue. International Agency of Research on Cancer 2017; 4. überarbeitete Version.

Swolin B et al. A prospective long-term cytogenetic study in polycythemia vera in relation to treatment and clinical course. Blood 1988;72(2):386-395.

Swolin B et al. Therapy-related patterns of cytogenetic abnormalities in acute myeloid leukemia and myelodysplastic syndrome post polycythemia vera: single center experience and review of literature. Annals of Hematology 2008;87(6):467-474.

Tang G et al. Characteristics and clinical significance of cytogenetic abnormalities in polycythemia vera. Haematologica 2017;102(9):1511-1518.

Tefferi A et al. Survival and prognosis among 1545 patients with contemporary polycythemia vera: an international study. Leukemia 2013;27(9):1874-1881.

Tefferi A et al. Targeted deep sequencing in polycythemia vera and essential thrombocythemia. Blood Advances 2016;1:21-30.

Tefferi A et al. (1) Primary myelofibrosis: 2019 update on diagnosis, risk-stratification and management. American Journal of Hematology 2018;93(12):1551-1560.

Tefferi A et al. (2) MIPSS70+ Version 2.0: Mutation and Karyotype-Enhanced International Prognostic Scoring System for Primary Myelofibrosis. J Clin Oncol 2018;36(17):1769-1770.

Tefferi A et al. Polycythemia vera and essential thrombocythemia: 2019 update on diagnosis, risk-stratification and management. American Journal of Hematology 2019;94:133–143.

Vannucchi AM et al. Polycythemia vera and essential thrombocythemia: algorithmic approach. Current Opinion of Hematology 2018;25(2):112-119.

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