Diagnostics

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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Classification
Diagnostics
Prognosis and therapy
Recommendation

Based on the current guidelines and the current state of research, there are different diagnostic recommendations for patients with polycythemia vera. We have summarized the most important information on classification and diagnostic methods at the MLL. In addition, we provide further literature and links on prognosis and therapy in polycythemia vera, so that you can inform yourself in more detail.

Polycythemia vera: Classification

Polycythemia vera (PV) is one of the myeloproliferative, BCR::ABL1-negative neoplasms (MPN). Usually, panmyelosis is present in PV. Nevertheless, PV is characterized by a predominant proliferation of erythrocytes, which is why secondary erythrocytosis (e.g., reactive polyglobulia due to stress, smoking, cardiac causes, and infections) should always be excluded for differential diagnosis. The PV variant with exclusive proliferation of the red cell line is called polycythemia vera rubra, but occurs very rarely. The subset of PV formerly referred to as the "masked" form of PV, which has lower hemoglobin and hematocrit values than the form originally defined in WHO 2008 (Barbui et al. 2014) is categorized as MPN, NOS according to the new WHO 2022.

Clinically, polycythemia vera is divided into 2 phases (WHO 2022):

Chronic phase (pre-polycythemic and polycythemic phase): Overproduction of erythrocytes and associated elevated hemoglobin and hematocrit levels
Late phase (post-PV myelofibrosis): Transition of the disease into secondary myelofibrosis

Table 1: WHO diagnostic criteria for polycythemia vera (WHO 2022)

According to the WHO classification, the diagnosis of polycythemia vera requires either all three major criteria or the first two major criteria and the minor criterion.

Major criteria:

Elevated hemoglobin concentration (♂: >16.5 g/dL, ♀: >16g/dL) or elevated hematocrit (♂: >49%*, ♀: >48%).
Bone marrow biopsy showing age-adjusted hypercellularity with trilineage growth (panmyelosis), including prominent erythroid, granulocytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes (differences in size)**.
Presence of JAK2 V617F or JAK2 exon 12 mutation

Minor criterion:

Subnormal serum erythropoietin level

* Hematocrit for diagnosis in the absence of a JAK2 mutation. A higher hematocrit target could be considered (e.g., 0.52) in men before further investigation may be required.

** Major criterion 2 (bone marrow biopsy) may not be required in patients with sustained absolute erythrocytosis (hemoglobin concentrations of >18.5 g/dL in men or >16.5 g/dL in women or hematocrit values of >0.555 in men or >0.495 in women), if major criterion 3 and the minor criterion are present.

The clinical differentiation of PV within the myeloproliferative neoplasms is based, among other things, on the detection of clonal erythrocytosis according to the WHO classification 2022 (Table 1). Currently, however, there are no specific disease markers, molecular or otherwise, that unequivocally diagnose polycythemia vera, which is why a diagnosis should always be made on the basis of a combination of clinical and bone marrow histological findings.

After a median observation period of 10 years, the rate of leukemic transformation of PV patients is approximately 2.3-14.4%, and 5.5-18.7% after 15 years.

Table 2: Selected risk factors for leukemic transformation (WHO 2022)

Age

Leukocytosis

Leukocyte count ≥ 15 x 10 /L⁹
Leukocyte count > 13 x 10 /L⁹

Aberrant karyotype

Most common: del(20q), +9, +8, double anomalies, complex karyotype (≥3 aberrations involving chromosomes 1, 5, 7, and 9).

Cytoreductive agents

Pipobroman,³² P, chlorambucil

Mutations

SRSF2, IDH2, RUNX1
TP53
ASXL1, SRSF2; IDH1, IDH2
Acquisition of one or more additional mutations during the chronic phase

Polycythemia vera: Diagnostic methods

Cytomorphologic assessment in MPNs includes cellularity in the whole as well as in the individual hematopoietic series. It is also important to determine the blast percentage in order to distinguish acceleration (blast percentage 10-19%) or blast crisis (blast percentage >20%).

Characteristic changes in polycythemia vera include:

Marked hypercellularity
Granulopoiesis is increased but not left shifted
An increased and left shifted erythropoiesis
Megakaryocytes are mostly present proliferated as well as in loose clusters, nuclei are mostly hyperlobulated
Decreased iron storage in the KM

Chromosomal aberrations are detected in 14-20% of patients with polycythemia vera at initial diagnosis (Swolin et al. 1988, Gangat et al. 2008, Sever et al. 2013) and in approximately 20% in a chronic phase. In the advanced and accelerated phases, respectively, cytogenetic changes are seen in 45% and 90% of patients (Tang et al. 2017). At initial diagnosis, deletions in the long arm of chromosome 20 (del(20q)), trisomy 8 (+8), and trisomy 9 (+9) are most commonly observed. Furthermore, deletions in the long arm of chromosome 13 (del(13q)), in the short arm of chromosome 12 (del(12p)), and gains of material of the long arm of chromosome 1 (+1q) were described (Reilly 2008, Swolin et al. 2008, Sever et al. 2013, Cerquozzi & Tefferi 2015, Swerdlow et al. 2017, WHO 2022). Recent studies showed that an aberrant karyotype increases the risk for progression and overall worse outcome (Dingli et al. 2006, Tefferi et al. 2013, Tang et al. 2017). As the disease progressed, complex karyotypes were observed increasingly, which mainly showed chromosomal alterations in the long arm of a chromosome 5 (del(5q)), aberrations of chromosome 7 (-7/del(7q)), chromosome 17 (-17/del(17p)/add(17p)), and a monosomy 18 (-18) (Tang et al. 2017). However, none of these aberrations are specific to PV; they also occur in other MPNs and also in MDS and AML (Swerdlow et al. 2017).

FISH is predominantly used in addition to cytogenetics to provide quantitative information on clone size and to obtain a marker suitable for progression. However, this method can only be used for specific questions, such as the exclusion of a BCR::ABL1 rearrangement within the myeloproliferative neoplasms, and will therefore not be able to replace the classical chromosome analysis. Detection of cytogenetic alterations typical for polycythemia vera can be performed on both blood and bone marrow smears.

JAK2 V617F mutations in polycythemia vera

In about 95% of patients with PV, a V617F mutation can be detected in exon 14 of the Janus kinase 2 (JAK2) gene. If this cannot be detected, mutations in exon 12 of the JAK2 gene (3%) can be found in a further proportion of patients (see Table 3). JAK2 mutations are thus found in the largest proportion of PV patients (>95%), but are not specific for PV (WHO 2022). The other "driver" mutations in CALR and MPL, which are commonly diagnosed in other MPNs such as PMF or ET, tend not to occur in PV. JAK2, CALR and MPL are primarily determined in the routine diagnostic program. If these are negative, other "non-driver" mutations can be investigated (see Table 3).

Mutations in the ASXL1, IDH2 as well as SRSF2 genes represent relevant alterations for an unfavorable prognosis of patients (WHO 2022).

Table 3: Frequency of different mutations in polycythemia vera (Luque Paz et al. 2023)

Gen-Mutation	Häufigkeit (%)	Clinical effect according to WHO 2022
JAK2 V617F (Exon 14)	>95
JAK2 Exon 12	3
MPL	0
CALR	0
TET2	10-20
DNMT3A	5-10
IDH1/2	1-2	Impact on leukemic transformation, progression to post-PV myelofibrosis, survival
ASXL1	2-7	Influence on leukemic transformation, survival
EZH2	1-2
SRSF2	<2	Influence on leukemic transformation, survival
SF3B1	2-3
U2AF1	<2
RUNX1	<2	Influence on leukemic transformation
TP53	<2	Influence on leukemic transformation

Polycythemia vera: Prognosis and therapy

Due to strong variation, risk factors affecting survival expectancy should be assessed individually for each patient to the greatest extent possible. Untreated patients with polycythemia vera show an extremely shortened life expectancy (1.5 years) compared to that of treated patients (median survival between 14 and 19 years) (Tefferi & Barbui 2019).

The risk stratification for the therapy decision is based on the risk of thrombosis. The risk factors for this are age (≥ 60 years) and previous thromboembolism. Prognostic models are predominantly based on clinical data (Bonicelli et al. 2013, Tefferi et al. 2013, Vannucchi et al. 2018) However, because patients are primarily at risk for disease progression toward secondary myelofibrosis (SMF), a "Myelofibrosis Secondary to PV and ET-Prognostic Model (MYSEC-PM)" was developed by Passamonti's group (Passamonti et al. 2017). An aberrant karyotype and mutations in the SRSF2 gene, among others, are included in the MIPSS-PV (mutation-enhanced international prognostic score) in the calculation of risk (Tefferi et al. 2020, Tefferi & Barbui 2023). High JAK2 V617F variant allele frequency (VAF) is also considered a prognostic negative factor for overall survival (Kanduła et al. 2023), which also influences thrombosis risk and progression (Zhang et al. 2020, Moliterno et al. 2023, Tefferi & Barbui 2023).

The main goals of therapy are to reduce the risk of thrombosis, to control clinical symptoms, and to delay or prevent myelofibrosis, MDS, or acute leukemia. An overview of therapeutic options is provided in the Onkopedia guideline Polycythemia vera. In a summary by How et al. there is also a review of selected therapeutics already approved or in clinical development for MPNs (How et al. 2023).

Polycythemia vera: Recommendation

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

Barbui T et al. Masked polycythemia vera diagnosed according to WHO and BCSH classification. Am J Hematol 2014;89(2):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. Br J Haematol 2013;160(2):251-254.

Cerquozzi S, Tefferi A. Blast transformation and fibrotic progression in polycythemia vera and essential thrombocythemia: a literature review of incidence and risk factors. Blood Cancer J 2015;5(11):e366.

Dingli D et al. Presence of unfavorable 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. Eur J Haematol 2008;80(3):197-200.

How J et al. Biology and therapeutic targeting of molecular mechanisms in MPNs. Blood 2023;141(16):1922-1933.

Kanduła Z et al. JAK2V617F variant allele frequency, non-driver mutations, single-nucleotide variants and polycythemia vera outcome. J Cancer Res Clin Oncol 2023;149(8):4789-4803.

Luque Paz D et al. Genetic basis and molecular profiling in myeloproliferative neoplasms. Blood 2023;141(16):1909-1921.

Moliterno AR et al. JAK2 V617F allele burden in polycythemia vera: burden of proof. Blood 2023;141(16):1934-1942.

Onkopedia Leitlinie „Polycythaemia Vera (PV)“ 2023; DGHO 2023.

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(12):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.

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

Swerdlow SH et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer (IARC) 2017; 4th.

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. Ann Hematol 2008;87(6):467-474.

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.

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

Tefferi A, Barbui T. Polycythemia vera and essential thrombocythemia: 2019 update on diagnosis, risk-stratification and management. Am J Hematol 2019;94(1):133-143.

Tefferi A, Barbui T. Polycythemia vera: 2024 update on diagnosis, risk-stratification, and management. Am J Hematol 2023.

Tefferi A et al. Mutation-enhanced international prognostic systems for essential thrombocythaemia and polycythaemia vera. Br J Haematol 2020;189(2):291-302.

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

Vannucchi AM et al. Polycythemia vera and essential thrombocythemia: algorithmic approach. Curr Opin Hematol 2018;25(2):112-119.

WHO Classification of Tumours Editorial Board. Haematolymphoid tumours [Internet; beta version ahead of print]. Lyon (France): International Agency for Research on Cancer; 2022. (WHO classification of tumours series, 5th ed.; vol. 11). Available from: https://tumourclassification.iarc.who.int/chapters/63.

Zhang Y et al. Thrombosis among 1537 patients with JAK2(V617F) -mutated myeloproliferative neoplasms: Risk factors and development of a predictive model. Cancer Med 2020;9(6):2096-2105.

Status: November 2023

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Polycythaemia vera (PV)

Polycythemia vera: Classification

Table 1: WHO diagnostic criteria for polycythemia vera (WHO 2022)

Table 2: Selected risk factors for leukemic transformation (WHO 2022)

Polycythemia vera: Diagnostic methods

Cytomorphology

Chromosome analysis

Fluorescence in situ hybridisation (FISH)

Molecular genetics

JAK2 V617F mutations in polycythemia vera

Table 3: Frequency of different mutations in polycythemia vera (Luque Paz et al. 2023)

Polycythemia vera: Prognosis and therapy

Polycythemia vera: Recommendation

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