Diagnostics

Primary myelofibrosis (PMF)

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Classification
Diagnostics
Prognosis
Recommendation

Based on the current guidelines and the current state of research, there are different diagnostic recommendations for patients with primary myelofibrosis. We have summarized the most important information on classification and diagnostic methods at the MLL. In addition, we provide links on prognosis and therapy in primary myelofibrosis.

Primary myelofibrosis: Classification

Primary myelofibrosis (PMF) is one of the myeloproliferative, BCR::ABL1 negative neoplasms (MPN). It is characterized by a predominant proliferation of megakaryocytes and granulocytes in the bone marrow and shows increasing reticulin and/or collagen fibrosis in advanced stages.

According to WHO classification 2022, PMF is divided into 2 stages (WHO 2022):

Primary myelofibrosis, prefibrotic
Primary myelofibrosis, fibrotic

The incidence of PMF is 0.44-1.5 / 100,000 person-years and occurs predominantly in the 60-70 years age range (WHO 2022).

Criteria for the diagnosis of primary myelofibrosis

According to the WHO classification, all 3 major criteria and at least 1 minor criterion of the diagnostic criteria must be met to establish the diagnosis of primary myelofibrosis (fibrotic stage).

Table 1: WHO diagnostic criteria for PMF in fibrotic stage (WHO 2022)

The diagnosis of overt PMF requires that all three major criteria and at least one minor criterion are met.

Major criteria:

Megakaryocytic proliferation and atypia, accompanied by reticulin and/or collagen fibrosis grades 2 or 3
WHO criteria for ET, PV, BCR::ABL1-positive CML, MDS, or other myeloproliferative neoplasms* are not met
JAK2, CALR, or MPL mutation

Presence of another clonal marker**

Absence of reactive myelofibrosis***

Minor criteria:

Presence of at least one of the following, confirmed in 2 consecutive determinations:

Anemia not attributed to a comorbid condition
Leukocytosis ≥ 11 x 10 /L⁹
Splenomegaly detected clinically and/or by imaging
Lactate dehydrogenase level above the upper limit of the institutional reference range
Leukoerythroblastosis

* Myeloproliferative neoplasms can be associated with monocytosis or they can develop it during the course of the disease; these cases may mimic chronic myelomonocytic leukaemia (CMML); in these rare instances, a history of MPN excludes CMML, whereas the presence of MPN features in the bone marrow and/or MPN-associated mutations (in JAK2, CALR, or MPL) tend to support the diagnosis of MPN with monocytosis rather than CMML.

** In the absence of any of the 3 major clonal mutations, a search for other mutations associated with myeloid neoplasms (e.g. ASXL1, EZH2, TET2, IDH1, IDH2, SRSF2, and SF3B1 mutations) may be of help in determining the clonal nature of the disease.

*** Bone marrow fibrosis secondary to infection, autoimmune disorder or another chronic inflammatory condition, hairy cell leukaemia or another lymphoid neoplasm, metastatic malignancy, or toxic (chronic) myelopathy.

Primary myelofibrosis: Diagnostic methods and their relevance

Cytomorphology and histology provide direction for guiding downstream diagnostics. Cytomorphologic assessment in MPNs involves cellularity in the whole as well as in individual hematopoietic series. It is also important to determine the blast percentage. In addition, for specific questions (suspected refractory anemia with ring sideroblasts and thrombocytosis; MDS/MPN-RS-T), iron staining is relevant.

If there is fibrosis of the bone marrow, e.g. in primary myelofibrosis (PMF), the cytomorphological assessability of the preparations is often limited ("punctio sicca").

However, the histology is decisive for the diagnosis as well as for the assessment of the degree of fibers and the bone marrow architecture and must always be performed in the case of suspected or confirmed MPN.

Cytogenetic aberrations occur in 30% of patients with primary myelofibrosis and are predominantly unbalanced events (WHO 2022). None of these aberrations are specific to primary myelofibrosis; they also occur in other MPNs and also in MDS (WHO 2022). The cytogenetic aberrations play an important role in risk stratification and therefore also influence the treatment decision for allogeneic stem cell transplantation, which is the only curative therapy (Tefferi et al. 2018 [3]).

Table 2: Cytogenetic risk stratification for PMF (Tefferi et al. 2018 [3])

Cytogenetic risk category	Specific aberrations
Favorable	Normal karyotype, single 20q-, single 13q-, single +9, single -Y, single aberration of a sex chromosome, single chromosome 1 translocation/duplication
Unfavorable	Single +8, single 7q-, single translocation (without involvement of chromosome 1), 2 aberrations (without aberration with very high risk), single/multiple 5q- aberrations, complex karyotype (without aberration with very high risk), monosomal karyotype (without aberration with very high risk), single aberrations not classified elsewhere
Very high risk	Single/multiple monosomy 7, single/multiple inv(3)/3q21 aberrations, single/multiple i(17q) aberrations, single/multiple 12p-/12p11.2 aberrations, single/multiple 11q-/11q23 aberrations, single/multiple autosomal trisomies other than +8 or +9 (e.g. +21, +19)

In case of punctio sicca, detection of cytogenetic alterations typical for primary myelofibrosis can be performed on a blood smear by FISH analysis.

JAK2 V617F mutations are common in primary myelofibrosis

Molecularly, a JAK2 mutation is detected in approximately 60% of patients with primary myelofibrosis (Luque Paz et al. 2023).

CALR mutations occur in about 25-35% of PMF patients and activating point mutations of the MPL gene in about 5-10%. Patients without any of the three diagnostic mutations ("triple-negative") have an increased risk of developing AML (Rumi et al. 2014, Tefferi et al. 2014 [1]). Type 1 CALR mutations occur significantly more frequently in PMF than type 2 mutations and show a more favorable prognosis than other mutations in this context (WHO 2022).

In addition, more than half of MPN patients are found to have mutations in other genes that play a role in the (initial) development of the disease. Among the most common are mutations in TET2 (10-15% of MPN patients), ASXL1 (5-10%), and DNMT3A (5-10%). Mutations in splicing regulators (SRSF2, SF3B1, U2AF1, ZRSR2) also occur with a lower prevalence, as well as mutations in other regulators of chromatin structure, epigenetic functions, and cellular signaling (e.g., EZH2, IDH1, IDH2, CBL, KRAS, NRAS, STAG2, TP53). These additional mutations are more common in myelofibrosis and blast phase than in pre-PMF, PV, and ET. Some of these mutations are known to be associated with worse prognostic risk (e.g., EZH2, IDH1, IDH2, SRSF2, and ASXL1 mutations in PMF). Mutation of TP53 is associated with leukemic transformation and also characterizes a disease category with a particularly unfavorable prognosis (WHO 2022).

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

Gene mutation	Frequency (%)	Clinical effect
JAK2	60	WHO diagnostic criterion
MPL	7-10	WHO diagnostic criterion
CALR	20-30	WHO diagnostic criterion
TET2	10-20	No known prognostic effect
DNMT3A	8-12	No known prognostic effect
IDH1/2	5-6	High molecular risk category
ASXL1	15-35	High molecular risk category
EZH2	7-10	High molecular risk category
NRAS	2-4	Unfavorable
KRAS	2	Unfavorable
SH2B3	2-4	Rare mutations in JAK2-negative MPNs
CBL	4	Unfavorable
SRSF2	6-14	High molecular risk category
SF3B1	5-7
U2AF1	7-10	Unfavorable
NFE2	3-5	Increased risk of leukemic transformation
RUNX1	2-3	Unfavorable
TP53	4-5	Unfavorable

Primary myelofibrosis: Prognosis and therapy

Among BCR::ABL1-negative myeloproliferative neoplasms, primary myelofibrosis has the most unfavorable course. There is both the risk of progression of the disease to the fibrotic stage and the risk of leukemic transformation. While the median overall survival is approximately 6-7 years (Passamonti & Mora 2023), individual clinical courses are highly heterogeneous. Prognosis is influenced by a variety of factors, which can be divided into clinical, cytogenetic, and molecular genetic factors. Especially for genetic alterations, the description of prognostically relevant factors continues to be the subject of research. With increasing knowledge, various prognostic scoring systems (~PSS) have been successively established or further developed (Table 4). An overview is also given in the Onkopedia guideline Primary Myelofibrosis.

Table 4: Prognostic scoring systems in PMF

Score	Prognostic factors
IPSS (Cervantes et al. 2009)	Clinical
DIPSS (Passamonti et al. 2010)	Clinical
DIPSS Plus (Gangat et al. 2011)	Clinical, karyotype
GPSS (Tefferi et al. 2014 [1])	Karyotype, mutations
MIPSS (Vannucchi et al. 2014)	Clinical, mutations
GIPSS (Tefferi et al. 2018 [2])	Karyotype, mutations
MIPSS70 (Guglielmelli et al. 2018)	Clinical, mutations
MIPSS70+ (Guglielmelli et al. 2018)	Clinical, karyotype, mutations
MIPSS70+ Version 2.0 (Tefferi et al. 2018 [1])	Clinical, karyotype, mutations

An overview of selected therapeutics already approved or in clinical development for MPNs is provided in a summary by How et al. (How et al. 2023).

Primary myelofibrosis: Recommendation

In addition to clinical and laboratory parameters, histological and cytomorphological examination of bone marrow and blood, cytogenetic analysis, and molecular genetic testing (JAK2 V617F mutation, if negative CALR, if negative MPL) are recommended. According to the WHO classification 2022, in primary myelofibrosis additional molecular genetic analyses should be performed if necessary (ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2, SF3B1).

Cervantes F et al. New prognostic scoring system for primary myelofibrosis based on a study of the International Working Group for Myelofibrosis Research and Treatment. Blood 2009;113(13):2895-2901.

Gangat N et al. DIPSS plus: a refined Dynamic International Prognostic Scoring System for primary myelofibrosis that incorporates prognostic information from karyotype, platelet count, and transfusion status. J Clin Oncol 2011;29(4):392-397.

Guglielmelli P et al. MIPSS70: Mutation-Enhanced International Prognostic Score System for Transplantation-Age Patients With Primary Myelofibrosis. J Clin Oncol 2018;36(4):310-318.

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

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

Onkopedia Leitlinie „Primäre Myelofibrose (PMF)“ 2021; DGHO 2021.

Passamonti F et al. A dynamic prognostic model to predict survival in primary myelofibrosis: a study by the IWG-MRT (International Working Group for Myeloproliferative Neoplasms Research and Treatment). Blood 2010;115(9):1703-1708.

Passamonti F, Mora B. Myelofibrosis. Blood 2023;141(16):1954-1970.

Rumi E et al. Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis. Blood 2014;124(7):1062-1069.

Tefferi A et al. Integration of Mutations and Karyotype Towards a Genetics-Based Prognostic Scoring System (GPSS) for Primary Myelofibrosis. Blood 2014 [1];124(21):406a.

Tefferi A et al. MIPSS70+ Version 2.0: Mutation and Karyotype-Enhanced International Prognostic Scoring System for Primary Myelofibrosis. J Clin Oncol 2018 [1];36(17):1769-1770.

Tefferi A et al. GIPSS: genetically inspired prognostic scoring system for primary myelofibrosis. Leukemia 2018 [2];32(7):1631-1642.

Tefferi A et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia 2014 [1];28(7):1472-1477.

Tefferi A et al. Revised cytogenetic risk stratification in primary myelofibrosis: analysis based on 1002 informative patients. Leukemia 2018 [3];32(5):1189-1199.

Vannucchi AM et al. Mutation-Enhanced International Prognostic Scoring System (MIPSS) for Primary Myelofibrosis: An AGIMM & IWG-MRT Project. Blood 2014;124(21):405a.

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.

Status: November 2023

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Primary myelofibrosis (PMF)

Primary myelofibrosis: Classification

Criteria for the diagnosis of primary myelofibrosis

Table 1: WHO diagnostic criteria for PMF in fibrotic stage (WHO 2022)

Primary myelofibrosis: Diagnostic methods and their relevance

Cytomorphology

Chromosome analysis

Table 2: Cytogenetic risk stratification for PMF (Tefferi et al. 2018 [3])

Fluorescence in situ hybridisation (FISH)

Molecular genetics

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

Primary myelofibrosis: Prognosis and therapy

Table 4: Prognostic scoring systems in PMF

Primary myelofibrosis: Recommendation

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