Virchows Archiv (2023) 482:53–68

https://doi.org/10.1007/s00428-022-03480-8

REVIEW

International Consensus Classification of myeloid and lymphoid

neoplasms: myeloproliferative neoplasms
Umberto Gianelli1 · Jürgen Thiele2 · Attilio Orazi3 · Naseema Gangat4 · Alessandro M. Vannucchi5 · Ayalew Tefferi4 ·
Hans Michael Kvasnicka6

Received: 2 September 2022 / Revised: 11 December 2022 / Accepted: 16 December 2022 / Published online: 29 December 2022
© The Author(s) 2022

Abstract
The recently published International Consensus Classification (ICC) of myeloid neoplasms summarized the results of an in-
depth effort by pathologists, oncologists, and geneticists aimed to update the 2017 World Health Organization classification
system for hematopoietic tumors. Along these lines, several important modifications were implemented in the classification
of myeloproliferative neoplasms (MPNs). For chronic myeloid leukemia, BCR::ABL1-positive, the definition of accelerated
and blast phase was simplified, and in the BCR::ABL1-negative MPNs, the classification was slightly updated to improve
diagnostic specificity with a more detailed and better validated morphologic approach and the recommendation of more
sensitive molecular techniques to capture in particular early stage diseases. In this regard, high sensitive single target (RT-
qPCR, ddPCR) or multi-target next-generation sequencing assays with a minimal sensitivity of VAF 1% are now important
for a proper diagnostic identification of MPN cases with low allelic frequencies at initial presentation. This review discusses
the updated diagnostic criteria of MPN according to the ICC, particularly by highlighting the new concepts and how they
can be applied in clinical settings to obtain an appropriate prognostic relevant diagnosis.

Keywords International Consensus Classification · Myeloid and lymphoid neoplasms: Myeloproliferative neoplasms

Introduction Association of Haematopathology (EAHP) and Society

for Hematopathology (SH) during the 20th Meeting of the
The International Consensus Classification (ICC) of myeloid European Association of Haematopathology (Virtual, April
and lymphoid neoplasms [1] represents the results of an in- 2021) and the collaborative work of hematopathologists,
depth discussion by hematopathologists from the European hematologists, and molecular biologists during the Clinical
Advisory Committee held in Chicago (September 2021) to
update the current classification of myeloid neoplasms [2].
* Hans Michael Kvasnicka According to the ICC guidelines, the category of mye-
hm.kvasnicka@patho-uwh.de
loproliferative neoplasms (MPN) include BCR::ABL1-
1
University of Milan, Department of Health Sciences and positive chronic myeloid leukemia (CML), essential
S.C. Anatomia Patologica, ASST Santi Paolo e Carlo, Milan, thrombocythemia (ET), primary myelofibrosis (PMF), and
Italy polycythemia vera (PV) as well as chronic neutrophilic leu-
2
Institute of Pathology, University of Cologne, Cologne, kemia (CNL) and chronic eosinophilic leukemia (CEL).
Germany In the CML group, the main effort of the ICC resulted in a
3
Department of Pathology, Texas Tech University Health simplified definition of accelerated and blast phase (AP and
Sciences Center, El Paso, TX, USA BP), while in the other MPN subtypes, reduction of diag-
4
Mayo Clinic, Rochester, MN, USA nostic uncertainty, especially in initial disease stages, and
5
CRIMM‑Centro Ricerca e Innovazione delle Malattie the identification of specific molecular lesions were in focus
Mieloproliferative, Azienda Ospedaliera‑Universitaria to optimize clinical management of patients. In all MPN
Careggi, Department of Experimental and Clinical Medicine, subtypes, high sensitive single target (RT-qPCR, ddPCR)
University of Florence, Florence, Italy
or multi-target panel/next-generation sequencing (NGS)
6
University Clinic Wuppertal, University of Witten/Herdecke, assays with a minimal sensitivity of 1% are recommended
Wuppertal, Germany

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to identify molecular alteration even with low variant allelic bone marrow or peripheral blood blasts, peripheral blood
frequencies (VAF) at initial diagnosis. basophilia > 20%, or the identification of additional clonal
This review aims to discuss diagnostic criteria of MPN cytogenetic abnormalities. Previous criteria for AP that have
according to the ICC, particularly by highlighting and elabo- been included in the WHO 2017 definition like thrombocy-
rating new aspects and their application for diagnosis. topenia (≤ 100 × ­109/L) unrelated to therapy or unrespon-
sive thrombocytosis (> 1000 ×  ­109/L) and/or splenomegaly
to therapy have been discarded by the ICC CML working
Chronic myeloid leukemia, group. Furthermore, the failure to achieve complete hema-
BCR::ABL1‑positive tological response or resistance to sequential tyrosine kinase
inhibitors, or occurrence of > 2 mutations on BCR::ABL
Clinical features during treatment, has also been eliminated in the definition
of AP. CML-BP is characterized by 20% or more myeloid
Incidence of chronic myeloid leukemia, BCR::ABL1–posi- blasts or extramedullary myeloid sarcoma. Importantly,
tive, (CML) in the general population accounts for 1–2 the presence of > 5% lymphoid blasts in peripheral blood
cases per 100.00 adults and about 15% of newly diagnosed or bone marrow is defining lymphoblastic crisis and thus
cases of leukemia in adults [3]. Since the introduction of should prompt further laboratory and genetic studies [5–7].
tyrosine kinase inhibitors (TKI) in 2000 annual mortality of In established AP or BP, or in patients with clinical features
CML has decreased from 10–20% to 1–2%, with significant suggesting disease progression (e.g., progressive splenomeg-
increase of the prevalence in well-developed countries and aly), bone marrow evaluation is recommended. Noteworthy,
an improvement of the 10-year survival rate from approxi- mild increase in bone marrow fibrosis (MF-1), even at ini-
mately 20% to 80–90% [4]. However, in cases with ineffec- tial diagnosis, correlates with a decreased major molecular
tive therapy, CML may evolve into AP and BP (myeloid in response (MMR) rate in the first year of TKI therapy [8, 9].
about 60% of the cases, lymphoid in 30%, and megakaryo-
cytic or undifferentiated in 10%). Importantly, leukemic evo- Morphology
lution can present also without a previous AP. Diagnosis of
chronic phase CML (CP-CML) which is mainly based on In CP-CML, peripheral blood displays leukocytosis (median
the detection of the BCR::ABL1 rearrangement remained value: 80 × 1­ 09/L) with neutrophils in various stages of
unchanged, while the diagnostic criteria for AP and BP have maturation and increase of myelocytes and segmented neu-
been simplified by the ICC CML working group. The ICC trophils without significant dysplasia [6]. Absolute baso-
guidelines have maintained a blast percentage threshold of philia and eosinophilia are frequent findings. It should be
10–19% and at least 20% in the blood or BM to establish the noted that some patients lack significant leukocytosis and
diagnosis of AP and BP, respectively. Of note, other clas- present with a sustained thrombocytosis mimicking ET at
sification systems which include the International Blood and initial diagnosis. However, the majority of patients is diag-
Marrow Transplant Registry (IBMTR), M.D. Anderson Can- nosed in the setting of a persistent unexplained leukocy-
cer Center (MDACC), and the European LeukemiaNet have tosis, and the diagnosis of CP-CML is established by the
defined a higher blast threshold of more than 30% and are characteristic Philadelphia (Ph) chromosome abnormality
more frequently used as eligibility criteria in clinical trials. t(9; 22)(q34;q11), assessed either by routine cytogenetics or
According to the ICC criteria, AP is defined by 10–19% the detection of a BCR::ABL1 abnormality by fluorescence

Table 1  Diagnostic criteria for accelerated and blast phase chronic myeloid, BCR::ABL1–positive (CML) according to the International Con-
sensus ­Classification1

Accelerated Bone marrow or peripheral blood blasts 10–19% Blast phase Bone marrow or peripheral blood myeloid blasts ≥ 20%
phase
Peripheral blood basophils ≥ 20% Myeloid ­sarcomab
Presence of additional clonal cytogenetic abnormal- Bone marrow or peripheral blood lymphoid blasts >
ity in Ph+ cells (ACA)a 5% is consistent with lymphoblastic ­crisisc

Ph, Philadelphia chromosome

a
Second Ph, trisomy 8, isochromosome 17q, trisomy 19, complex karyotype ≥ 3 cytogenetic abnormalities, or abnormalities of 3q26.2
b
Extramedullary blast proliferation
c
Immunophenotypic analysis is required to confirm lymphoid lineage

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Fig. 1  Essential Thrombocythemia is characterized by a normocellu- hyperlobulated nuclei. Erythropoietic and granulopoietic series do
lar marrow with increased number of megakaryocytes forming loose not show significant abnormalities. Reticulin fibrosis is usually not
clusters and rarely dense clusters, with large to giant elements and increased (MF-0)

in situ hybridization or molecular studies. Although a bone changes in the granulocytic precursors and megakaryocytes
marrow biopsy is not required at diagnosis, baseline evalu- (i.e., myelodysplasia-like micro-megakaryocytes) together
ation of the hematopoietic series and of the grade of bone with an accumulation of reticulin/collagen fibers.
marrow fibrosis by reticulin staining can be prognostically
informative. In CP, the bone marrow is hypercellular for the Genetic profile
patient’s age with marked granulocytic proliferation with a
left-shift like in the peripheral blood, decreased erythroid In 95% of CML cases, the characteristic t(9;22)(q34.1;q11.2)
precursors, and an increased number of small megakaryo- translocation defined as Philadelphia chromosome is pre-
cytes (in about 40–50% of the cases) with hypolobulated sent. This translocation is responsible for the fusion gene
nuclei (“dwarf” megakaryocytes). Blasts usually account for BCR::ABL1 and the consequently chimeric protein p210.
less than 5% [10, 11]. Eosinophils and basophils are usually Different breakpoints and rearrangements can result in about
increased in number, and pseudo-Gaucher histocytes may 1% of patients in a shorter p190 oncoprotein and in 2–5%
be observed. Of note, cases carrying the p230 fusion protein of patients in a p210 variant or p230 transcript which usu-
often show a marked neutrophilic maturation and thrombo- ally is associated with a more indolent behavior [12]. Most
cytosis, while those cases associated with a p190 fusion pro- of the patients show only the Ph chromosome throughout
tein may present with an increased number of mature mono- the chronic phase. During CML progression to AP and BP,
cytes mimicking chronic myelomonocytic leukemia. In AP, secondary chromosomal abnormalities can be detected, most
the increased blast count can be associated with dysplastic commonly +8 (34% of cases with additional changes), +Ph

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Table 2  Diagnostic criteria for essential thrombocythemia and post-essential thrombocythemia myelofibrosis (post-ET MF) according to the
International Consensus ­Classification1
ET Post-ET MF

Major criteria 1. Platelet count ≥ 450 × 1­ 09/L Required criteria 1. Previous established diagnosis of ET
2. Bone marrow biopsy showing proliferation 2. Bone marrow fibrosis of grade 2 or 3 (MF-2 or
mainly of the megakaryocytic lineage, with MF-3)
increased numbers of enlarged, mature mega-
karyocytes with hyperlobulated staghorn-
like nuclei, infrequently dense ­clustersa; no
significant increase or left shift in neutrophil
granulopoiesis or erythropoiesis; no relevant
BM ­f ibrosisb
3. Diagnostic criteria for BCR::ABL1 positive Additional criteria Anemia (i.e., below the reference range given age,
chronic myeloid leukemia, polycythemia vera, sex, and altitude considerations) and a > 2 g/dL
primary myelofibrosis, or other myeloid neo- decrease from baseline hemoglobin concentration
plasms are not met
4. JAK2, CALR, or MPL ­mutationc Leukoerythroblastosis
Minor criteria 1. Presence of a clonal ­markerd or absence of Increase in palpable splenomegaly of > 5 cm
evidence of reactive ­thrombocytosise from baseline or the development of a newly
palpable splenomegaly
Elevated lactate dehydrogenase level above the
reference range
Development of any 2 (or all 3) of the following
constitutional symptoms: >10% weight loss in 6
months, night sweats, unexplained fever (> 37.5 °C)

The diagnosis of ET requires either all major criteria or the first 3 major criteria plus the minor criteria. The diagnosis of post-ET MF is estab-
lished by the two required criteria and at least two additional criteria
a
Three or more megakaryocytes lying adjacent without other BM cells in between; in most of these rare clusters < 6 megakaryocytes may be
observed, increase in huge clusters (> 6 cells) accompanied by granulocytic proliferation is a morphological hallmark of pre-PMF
b
Very rarely a minor increase in reticulin fibers may occur at initial diagnosis (MF-1)
c
It is recommended to use highly sensitive assays for JAK2V617F (sensitivity level < 1%) and CALR and MPL (sensitivity level 1–3%)—in
negative cases, consider a search for non-canonical JAK2 and MPL mutations
d
Assessed by cytogenetics or sensitive NGS techniques
e
Reactive causes of thrombocytosis include a variety of underlying conditions like iron deficiency, chronic infection, chronic inflammatory dis-
ease, medication, neoplasia, or history of splenectomy

(30%), i(17q) (20%), +19 (13%), -Y (8% of males), +21 Essential thrombocythemia
(7%), +17 (5%), and monosomy 7 (5%), which are often
associated with an unfavorable prognosis [13]. The acquisi- Clinical features
tion of major-route additional chromosomal abnormalities
on treatment is considered as hallmark of disease progres- ET incidence is estimated at 1.2 to 3.0 per 100,000 popu-
sion. About 30% of CML patients in CP with resistance lation per year [15] with a median age at diagnosis of 58
to first or second-generation of tyrosine kinase inhibitors years and a slight female predominance. More than 50%
(TKIs) harbor mutations in the BCR::ABL1 kinase domain. of the patients are asymptomatic and discovered inciden-
Additional mutations indicate a risk disease with higher rate tally with thrombocytosis (by definition > 450 × ­109/L).
of relapse on second- or subsequent line of therapy and fur- Symptoms are more frequently associated with thrombosis
ther acquisition of molecular abnormalities. Consequently, (ranging from transient ischemic attacks involving small
identification of BCR::ABL1 mutations in patients treated vessels to splanchnic vein thrombosis) or hemorrhages
with TKIs represents a biological hallmark of disease pro- (more frequently involving the gastrointestinal and res-
gression. In this context, NGS is highly sensitive to identify piratory tracts) [16, 17]. Mild splenomegaly can be seen
emerging resistant mutations even at the time of major or in about 15–20% of the cases, while hepatomegaly is rare.
deeper molecular responses [14]. Thrombohemorrhagic complications represent two of the
Diagnostic criteria for AP and BP CML are reported in main causes of morbidity and mortality in these patients
Table 1. [18, 19]. In a large cohort of patients, the rate of fatal

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Fig. 2  Early/pre-fibrotic PMF is characterized by a hypercellular mar- morphology. Erythropoiesis is often reduced, particularly in cases
row with pronounced granulopoiesis and increased number of mega- with increased reticulin fibrosis (MF-1)
karyocytes, usually forming dense clusters and displaying atypical

and non-fatal thrombotic events was 1.9% per patient/ Morphology

year [17]. Progression to post-ET MF has a cumulative
risk at 10 years ranging between 0.8 and 4.5%. Similar In the peripheral blood, the most frequent anomaly consists
to CML, in BCR::ABL1-negative MPN, progression to of thrombocytosis usually associated with anisocytosis of
AP is defined by the presence of 10 to 19% of periph- platelets. Bone marrow is normocellular for the patients’
eral blood or bone marrow blasts, while BP is defined by age, with only a few cases showing a mild hypercellular-
the presence of 20% or more blasts. Progression to AP ity (Fig. 1). Erythropoiesis, granulopoiesis, and myeloid/
at 10 years has been reported to range between 0.7 and erythroid ratio do not show significant abnormalities.
3% [20] and by strict adherence to the previous WHO Megakaryocytes are increased in number, usually large
criteria only between 0.7 and 1.9 % [21–23]. Moreover, to giant with hyper-lobulated nuclei and abundant mature
advanced age, extreme thrombocytosis, anemia, leukocy- cytoplasm. Frequently loose clusters can be observed, and
tosis, and additional mutations involving TP53 and EZH2 only very rarely they aggregate in dense clusters (usually
have been reported as risk factors for BP progression [24, small clusters with less than 6 cells). In these cases, the
25]. Median overall survival in ET patients ranges from differential diagnosis with pre-fibrotic PMF might be chal-
14.7 to about 21.8 years [21, 26]. lenging; however, identification of atypical megakaryocytes,

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Table 3  Diagnostic criteria for early/pre-fibrotic and overt primary myelofibrosis (PMF) according to the International Consensus C
­ lassification1
Early/pre-fibrotic—PMF Overt—PMF

Major criteria 1. Bone marrow biopsy showing megakaryocytic proliferation and a­ typiaa), bone mar- 1. Bone marrow biopsy showing megakaryocytic proliferation and ­atypiaa, accompanied
row fibrosis grade < 2, increased age-adjusted BM cellularity, granulocytic prolifera- by reticulin and/or collagen fibrosis grades 2 or 3
tion, and (often) decreased erythropoiesis
2. JAK2, CALR, or MPL ­mutationb or presence of another clonal ­markerc or absence of 2. JAK2, CALR, or MPL ­mutationb or presence of another clonal ­markerc or absence of
reactive bone marrow reticulin ­fibrosisd reactive bone marrow reticulin ­fibrosisd
3. Diagnostic criteria for BCR::ABL1 positive chronic myeloid leukemia, polycythemia 3. Diagnostic criteria for BCR::ABL1 positive chronic myeloid leukemia, polycythemia
vera, essential thrombocythemia, myelodysplastic syndromes, or other myeloid vera, essential thrombocythemia, myelodysplastic syndromes, or other myeloid
­neoplasmse are not met ­neoplasmse are not met
Minor criteria 1. Anemia not attributed to a comorbid condition 1. Anemia not attributed to a comorbid condition
2. Leukocytosis ≥ 11 × 1­ 09/L 2. Leukocytosis ≥ 11 × ­109/L
3. Palpable splenomegaly 3. Palpable splenomegaly
4. Lactate dehydrogenase level above the reference range 4. Lactate dehydrogenase level above the reference range
5. Leukoerythroblastosis

The diagnosis of pre-PMF or overt-PMF requires all 3 major criteria and at least 1 minor criterion confirmed in 2 consecutive determinations
a
Morphology of megakaryocytes in pre-PMF and overt PMF usually demonstrates a higher degree of megakaryocytic atypia than in any other MPN-subtype; distinctive features of megakaryo-
cytes include small to giant megakaryocytes with a prevalence of severe maturation defects (cloud-like, hypolobulated and hyperchromatic nuclei) and presence of abnormal large dense clusters
(mostly > 6 megakaryocytes lying strictly adjacent)
b
It is recommended to use highly sensitive assays for JAK2 V617F (sensitivity level < 1%) and CALR and MPL (sensitivity level 1−3%)—in negative cases, consider searching for non-canonical
JAK2 and MPL mutations.
c
Assessed by cytogenetics or sensitive NGS techniques; detection of mutations associated with myeloid neoplasms (e.g., ASXL1, EZH2, IDH1, IDH2, SF3B1, SRSF2, and TET2 mutations) sup-
ports the clonal nature of the disease
d
Minimal reticulin fibrosis (grade 1) secondary to infection, autoimmune disorder or other chronic inflammatory conditions, hairy cell leukemia or another lymphoid neoplasm, metastatic
malignancy, or toxic (chronic) myelopathies
e
Monocytosis can be present at diagnosis or develop during the course of PMF; in these cases, a history of MPN excludes CMML, whereas a higher variant allelic frequency for MPN-associ-
ated driver mutations is supporting the diagnosis of PMF with monocytosis rather than CMML
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Fig. 3  Polycythemia vera. Bone marrow is markedly hypercellular with panmyelosis. Megakaryocytes are increased in number can form loose
clusters and are typically polymorphic, showing variability in size but lack significant atypia. Reticulin fibrosis can be mildly increased (MF-1)

presence of granulocytic proliferation, and clinical features other side, CALR-positive patients are younger, more fre-
like increased LDH or splenomegaly support the diagnosis quently male, and characterized by higher platelet counts,
of pre-fibrotic PMF. In detail, megakaryocytic atypia in PMF lower hemoglobin level, lower leukocyte count, and lower
consists of nuclear and cytoplasmic abnormalities (increased incidence of thrombotic events. In this context, type 2 vs
nuclear/cytoplasmic ratio, irregular chromatin clumping, type 1 CALR mutations were associated with higher plate-
bulbous appearance, marked hyperchromasia). Myeloblasts let count [31, 32]. NGS analysis revealed that 53% of ET
are usually less than 5%, and a mild increase in reticulin fib- cases harbor one or more additional variants, other than
ers (grade 1) can be observed in less than 5% of patients at JAK2 V617F/CALR/MPL. The most frequent were TET2
initial diagnosis [27–29]. and ASXL1 [33]. Adverse variants for decreased over-
all, leukemia- or fibrosis-free survival included SH2B3,
Genetic profile SF3B1, U2AF1, TP53, IDH2, and EZH2. Overall survival
is impacted by SF3B1/SRSF2 mutations, whereas U2AF1
A JAK2V617F driver mutation can be found in about 60% and SF3B1 mutations may affect myelofibrosis-free sur-
of ET cases, calreticulin (CALR), and MPL mutations in vival and TP53 mutations predicted leukemic transforma-
about 20% and 3%, while only a small subgroup of patients tion. In this regard, assessment of MPN drivers and high
presents without one of these driver mutations (so-called molecular risk (HMR) mutations allow the calculation of
“wild-type”) [21, 30]. JAK2V617F has been associated a mutation-enhanced international prognostic system [34].
with an increased risk of thrombosis and a lower risk of Diagnostic criteria for the diagnosis of ET according to
myelofibrotic progression, i.e., post-ET MF [22]. On the the ICC are reported in Table 2.

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Table 4  Diagnostic criteria for polycythemia vera (PV) and post polycythemia vera myelofibrosis (post-PV MF) according to the International
Consensus ­Classification1
PV Post-PV MF

Major criteria 1. Elevated hemoglobin concentration or elevated Required criteria 1. Previous established diagnosis of PV
hematocrit or increased red blood cell ­massa
2. Bone marrow biopsy showing age-adjusted 2. Bone marrow fibrosis of grade 2 or 3
hypercellularity with trilineage proliferation
(panmyelosis), including prominent erythroid,
granulocytic, and increase in pleomorphic,
mature megakaryocytes without a­ typiab
3. Presence of JAK2V617F or JAK2 exon 12
­mutationc
Minor criterion Subnormal serum erythropoietin level Additional criteria 1. Anemia (i.e., below the reference range given
age, sex, and altitude considerations) or sustained
loss of requirement of either phlebotomy (in the
absence of cytoreductive therapy) or cytoreductive
treatment for erythrocytosis
2. Leukoerythroblastosis
3. Increase in palpable splenomegaly of > 5 cm from
baseline or the development of a newly palpable
splenomegaly
4. Lactate dehydrogenase level above the reference
range

The diagnosis of PV requires all 3 major criteria or the first two major criteria plus the minor criterion. The diagnosis of post-PV MF is estab-
lished by all two required criteria and at least two additional criteria
a
Diagnostic thresholds: hemoglobin: > 16.5 g/dL in men and > 16.0 g/dL in women; hematocrit: > 49% in men and > 48% in women; red blood
cell mass: > 25% above mean normal predicted value
b
A 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 and hematocrit values of > 55.5% in men or > 49.5% in women) and the presence of a JAK2 V617F or JAK2 exon
12 mutation
c
It is recommended to use highly sensitive assays for JAK2 V617F (sensitivity level < 1%)—in negative cases, consider searching for non-
canonical or atypical JAK2 mutations in exons 12–15

­ lassification1
Table 5  Diagnostic criteria for Myeloproliferative neoplasms, unclassifiable (MPN-U) according to the International Consensus C

1. Clinical and hematological features of a myeloproliferative neoplasm are ­presenta

2. JAK2, CALR, or MPL ­mutationb or presence of another clonal ­markerc
3. Diagnostic criteria for any other myeloproliferative neoplasm, myelodysplastic syndrome, myelodysplastic/myeloproliferative n­ eoplasmd,
or BCR::ABL1-positive chronic myeloid leukemia are not met

The diagnosis of MPN-U requires all 3 criteria

a
In cases presenting with BM fibrosis, reactive causes must be excluded, in particular BM fibrosis secondary to infection, autoimmune disor-
der or another chronic inflammatory condition, hairy cell leukemia or another lymphoid neoplasm, metastatic malignancy, or toxic (chronic)
myelopathy
b
It is recommended to use highly sensitive assays for JAK2V617F (sensitivity level < 1%) and CALR and MPL (sensitivity level 1–3%)—in
negative cases, consider searching for non-canonical JAK2 and MPL mutations
c
Assessed by cytogenetics or sensitive NGS techniques; detection of mutations associated with myeloid neoplasms (e.g., ASXL1, EZH2, IDH1,
IDH2, SF3B1, SRSF2, and TET2 mutations) supports the clonal nature of the disease
d
In cases presenting with myelodysplastic features, effects of any previous treatment, severe comorbidity, and changes during the natural pro-
gression of the disease process must be carefully excluded

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Fig. 4  Chronic neutrophilic leukemia. Bone marrow is markedly cytes. Megakaryocytes can be increased in number with mature mor-
hypercellular for the patient’s age, with hyperplastic granulopoiesis phology. Myeloblasts are usually less than 5% (CD34) (courtesy of E.
and increased number of metamyelocytes and segmented granulo- Sabattini Bologna, Italy)

Primary myelofibrosis centers of excellence, the incidence of pre-PMF in cases

originally diagnosed as ET may be calculated between 14
The ICC guidelines aim to increase diagnostic specificity, and 18% [21, 38, 39]. Approximately, 30–40% of pre-PMF
especially in initial/early cases of MPN presenting with patients are asymptomatic at diagnosis, but reveal an abnor-
thrombocytosis. Over the last years, several studies clearly mal CBC, usually slight anemia, or leukocytosis and less
confirmed clinical, morphological, and molecular differ- commonly gross splenomegaly. Thrombocytosis clinically
ences between the prefibrotic stage of PMF and ET, and mimicking ET is one of the most common and challenging
therefore, the definition of a prefibrotic stage as distinct dis- presentation in pre-PMF. Rarely, unexplained leukoeryth-
ease category within the MPN subtypes has been maintained roblastosis or an increased lactate dehydrogenase (LDH)
[26, 35–37]. level prompts the initial diagnosis. Compared to overt PMF,
patients with pre-PMF are often younger and present with
Early/pre‑fibrotic primary myelofibrosis higher hemoglobin and platelet counts and minimal leuko-
cytosis [35]. Symptomatic cases reveal constitutional symp-
Clinical features toms like fatigue, weight loss, night sweats, and dyspnea.
Borderline to minimal splenomegaly represents a common
According to results from reclassification studies of BM finding (90% of the cases), while hepatomegaly of various
biopsies and corresponding clinical data to differentiate degree can be documented in about half of the patients. The
“true” ET from pre-PMF, after centralized evaluations by median survival in pre-PMF has been reported to range

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­ lassification1
Table 6  Diagnostic criteria for chronic neutrophilic leukemia (CNL) according to the International Consensus C

1. Peripheral blood white blood cell count ≥ 13 × ­109/La

Segmented neutrophils plus banded neutrophils constitute ≥ 80% of the white blood cells
No significant dysgranulopoiesis
Circulating blasts only rarely o­ bservedb
Monocyte count < 10% of all leukocytes
2. Hypercellular bone marrow with neutrophil granulocytes increased in percentage and absolute number, showing normal maturation
3. CSF3R T618I or another activating CSF3R mutation or persistent neutrophilia (≥ 3 months), splenomegaly, and no identifiable cause
of reactive neutrophilia including absence of a plasma cell neoplasm or, if a plasma cell neoplasm is present, demonstration of clonality of
myeloid cells by cytogenetic or molecular studies
4. Not meeting diagnostic criteria for BCR::ABL1-positive chronic myeloid leukemia, polycythemia vera, essential thrombocythemia, pri-
mary myelofibrosis, or of a myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions

The diagnosis of CNL requires all 4 criteria

a
≥ 25 × ­109/L in cases lacking CSF3R T618I or another activating CSF3R mutation
b
10–19% blasts in peripheral blood or bone marrow represent CNL in accelerated phase (AP); > 20% blasts represent blast phase (BP)

between 11 and 17 years, contrasting only 7 years for overt (increased nucleus/cytoplasmic ratio, abnormal chromatin
PMF. Reticulin fibrosis (MF-1) and anemia at initial diag- clumping, bulbous, and hyperchromatic nuclei) and form
nosis were identified as risk factors for progression from abnormal large dense clusters as morphological key feature
pre-PMF to overt disease stage. Furthermore, variables asso- (major criterion). These huge clusters are defined by more
ciated with BP evolution are age > 65 years, leukocytosis (> than 6 megakaryocytes lying strictly adjacent without other
15 × 1­ 09/L), and LDH ratio > 1.5 times the normal institu- bone marrow cells in between. It is important to underline
tional value and cytogenetic abnormalities [40]. that the presence of this abnormal morphological feature is
a morphological hallmark of pre-PMF and in general not
Morphology seen in other MPN subtypes, particularly ET. Therefore, in
cases clinically assigned as ET occurrence of large dense
In pre-PMF, peripheral blood shows a mild anisopoikilo- clusters (according to the ICC definition) should always
cytosis without leukoerythroblastosis. Bone marrow is prompt a critical reevaluation of diagnosis by inclusion of
characteristically hypercellular for the patient’s age with other important features like increased LDH level, leuko-
pronounced proliferation and left shifting of granulopoietic cytosis ≥ 11 × ­109/L, anemia not attributed to a comorbid
precursors and increased myeloid/erythroid ratio (Fig. 2). condition, and palpable splenomegaly (minor criteria). By
Megakaryocytes are increased in number and characterized definition, reticulin fibrosis is absent (MF-0) or mild (MF-1)
by polymorphisms (variation in size and shape) and atypia in pre-PMF. In some cases of ET, smaller dense clusters of

Table 7  Diagnostic criteria for chronic eosinophilic leukemia not otherwise specified (CEL, NOS) according to the International Consensus
­Classification1

1. Peripheral blood hypereosinophilia (eosinophil count ≥ 1.5 × ­109/L and eosinophils ≥ 10% of white blood cells)
2. Blasts constitute < 20% cells in peripheral blood and bone marrow, not meeting other diagnostic criteria for ­AMLa
3. No tyrosine kinase gene fusion including BCR::ABL1, other ABL1, PDGFRA, PDGFRB, FGFR1, JAK2, or FLT3 fusions
4. Not meeting criteria for other well-defined MPN; chronic myelomonocytic leukemia, or systemic ­mastocytosisb
5. Bone marrow shows increased cellularity with dysplastic megakaryocytes with or without dysplastic features in other lineages and often sig-
nificant fibrosis, associated with an eosinophilic infiltrate or increased blasts ≥ 5% in the bone marrow and/or ≥ 2% in the peripheral blood
6. Demonstration of a clonal cytogenetic abnormality and/or somatic mutation(s)c

The diagnosis of CEL requires all 6 criteria

a
AML with recurrent genetic abnormalities with < 20% blasts is excluded
b
Eosinophilia can be seen in association with systemic mastocytosis (SM). However, “true” CEL, NOS may occur as SM-AMN (systemic mas-
tocytosis with an associated myeloid malignancies)
c
In the absence of a clonal cytogenetic abnormality and/or somatic mutation(s) or increased blasts, bone marrow findings supportive of the diag-
nosis will suffice in the presence of persistent eosinophilia, provided other causes of eosinophilia having been excluded

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Virchows Archiv (2023) 482:53–68 63

megakaryocytes (< 6 cells) can be found and thus may be a manifestations more frequently include anemia, marked
source of diagnostic confusion. In these challenging cases, hepatosplenomegaly, constitutional symptoms (e.g., fatigue,
separation from pre-PMF should be based on the critical night sweats, fever), cachexia, pruritus, and thrombo-hemor-
evaluation of the complete histological pattern (including rhagic complications. The cumulative incidences of myeloid
immunohistochemistry for megakaryocytes) based on over- BP are reported as 11% at 5 years and 23% at 10 years.
all cellularity, myeloid/erythroid ratio, and morphology and Causes of death include leukemic progression that occurs in
histotopography of the megakaryocytes (i.e., dense clusters) approximately 20% of patients, but many patients also die
and stromal changes (i.e., bone marrow fibrosis, osteoscle- of comorbid conditions including cardiovascular events and
rosis) along with clinical data. consequences of cytopenia, including infection or bleeding.

Genetic profile
Morphology
In pre-PMF, abnormal cytogenetics is found in about 18%
Due to the deposition of reticulin and collagen fibers in
and unfavorable karyotypes in 4–8% of cases. Unfavorable
the overt stage, overall cellularity progressively decreases
abnormalities consist in complex karyotype (> 3 abnormali-
including a significant reduction of the erythroid compart-
ties), isolated +8, isolated −7/7q−, sole abnormalities like
ment. In the end stages of disease, the intertrabecular mar-
i(17q), −5/5q−, 12p−, 11q23 rearrangement or inv(3), and
row spaces can be occupied mainly by collagen fibers, with
an abnormal karyotype with abnormalities of chromosomes
scattered myeloid precursors and abnormal megakaryocytes
5, 7, 17, or 12p−. Incidence of JAK2V617F mutations is
which tend to be smaller and more dysmorphic than in early
very similar in pre-PMF and ET, ranging between 52–67%
disease stage. Increased micro-vessel density, with dilated
and 54–66%, respectively [26, 35]. There is no difference
and distorted sinusoids, intra-sinusoidal hematopoiesis, and
in the distribution of MPN driver mutations (JAK2V617F,
osteosclerosis is a common feature. Noteworthy, accurate
MPLW515x, and CALR) between pre-PMF and overt-PMF.
grading of bone marrow fibrosis has been confirmed by sev-
JAK2V617F mutation was found in 67.2% of pre-PMF and
eral groups to be prognostically informative in PMF [41, 42].
58.2% of overt PMF, CALR type 1 and type 2 in 12.2% and
Peripheral blood leuko-erythroblastosis and anisopoikilo-
5.8%, and 17.8% and 4.4% of pre-PMF and overt PMF,
cytosis (with tear-drop erythrocytes) correlate with the
respectively; MPLW515x-mutated patients were 4.7% and
increase of bone marrow fibrosis.
6.0% in the 2 cohorts. On the contrary, the high mutation
Progression to AP and BP in PMF is defined by the docu-
risk (HMR) category (any mutations in ASXL1, SRSF2,
mentation of 10–19% and 20% or more of peripheral blood
IDH1, IDH2, EZH2) is more frequently observed in overt
or bone marrow blasts, respectively. In the bone marrow
PMF [35]. The proportion of patients lacking any driver
biopsy, immunohistochemistry with CD34 can facilitate the
mutation (“triple negative PMF”) is similar between pre-
identification of increased blasts. Along these lines, iden-
PMF and overt PMF ranging between 10.1 and 13.6%. These
tification of progenitor clusters and/or their paratrabecu-
triple-negative cases belong to a subgroup with high risk of
lar localization has been shown to indicate early disease
leukemic transformation and very poor prognosis. Most of
progression.
these triple-negative cases present with thrombocytopenia
and only rarely with splenomegaly. A pronounced prolifera-
tion of the granulopoiesis as seen in pre-PMF is less likely Genetic profile
and dysplastic changes of the erythropoietic elements may
be observed. Furthermore, cytogenetics frequently reveals Cytogenetics abnormalities accumulate in overt-PMF and
a trisomy 8, and molecular analysis shows an enrichment in can be identified in 30–40% of patients. A number of chro-
high-risk mutations, which overall might trigger the detri- mosomal abnormalities have been associated with a worse
mental effect on prognosis. Due to their clinical, morpho- outcome, in particular those defined by the Dynamic Inter-
logical, and molecular overlap with the heterogenous group national Prognostic Scoring System-plus: complex karyo-
of myelodysplastic/myeloproliferative neoplasms (MDS/ type or single or two abnormalities including 8, 7/7q-,
MPN), these cases can pose a diagnostic challenge. i(17q), 5/5q-, 12p-, inv(3), or 11q23 rearrangement. More
recently, a three-tiered risk model has been proposed includ-
Overt primary myelofibrosis ing a “very high risk (VHR)”- single/multiple abnormali-
ties of -7, i(17q), inv(3)/3q21, 12p- /12p11.2, 11q-/11q23,
Clinical features or other autosomal trisomies not including +8/+9. In this
cytogenetically defined group of patients, a 5-year survival
Incidence of overt PMF accounts for about 0.5–1.5 patients rate of only 8% has been reported independent of clinically
× 100.00 population per year. In overt disease, clinical derived prognostic systems, the presence of driver and

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64 Virchows Archiv (2023) 482:53–68

non-driver mutations, contrasting a 45% survival rate for myelofibrotic transformed end stage, i.e., post-PV MF.
patients with “favorable” karyotype [43]. More than 80% of Noteworthy, a mild degree of reticulin fibrosis (MF-1)
patients with overt-PMF carry variants/mutations other than can be identified in about 20% of cases at initial diagnosis
JAK2/CALR/MPL, in particular high-risk mutations which and has been associated with an increased risk to develop
are associated with overall prognosis and leukemia-free sur- post-PV MF [49].
vival (ASXL1, SRSF2, IDH1/IDH2, EZH2) [35]. Diagnostic criteria for the definition of AP and BP in PV
Diagnostic criteria for the diagnosis of PMF according to are the same as those used in ET and PMF.
the ICC are reported in Table 3.
Genetic profile

JAK2 mutational frequencies in PV are estimated at 97% for

Polycythemia vera
JAK2V617F and 3% for other activating JAK2 mutations,
including mutations in exon 12. Patients carrying JAK2
Clinical features
exon 12 mutation usually present with predominant erythro-
poiesis, subnormal serum erythropoietin level, and younger
PV incidence ranges between 0.01 and 2.8 cases per 100.00
age at diagnosis but are prognostically similar to those with
per year. Clinically, increase of the red cell mass is mainly
JAK2V617F [51]. Increased allele burden does not affect
associated with major symptoms including hypertension,
survival or leukemic transformation in PV, while a higher
increase blood viscosity micro-circulatory symptoms, pru-
JAK2V617F mutant allele burden might be associated with
ritus, and venous or arterial thrombosis. The latter com-
pruritus and fibrotic transformation [52]. Molecular analysis
plication is seen in about 20% of cases as the first clinical
revealed in 53% of PV patients additional adverse variants
manifestation. Therefore, in the setting of splanchnic vein
(ASXL1, SRSF2, IDH2/EZH2) which correlate with inferior
thrombosis and Budd-Chiari syndrome, the differential diag-
survival (median, 7.7 vs 16.9 years). This effect was inde-
nosis of PV should always be considered. The cumulative
pendent of conventional prognostic models, and interest-
risk for leukemic transformation in PV has been reported
ingly, the number of mutations did not provide additional
as 2.3% at 10 years and 5.5% at 15 years [44]. Risk factors
prognostic information [34]. However, an abnormal karyo-
for leukemic progression include advanced age, leukocyto-
type has been reported in about 15–20% of patients with
sis, abnormal karyotype, and mutations involving SRSF2
PV and post-PV MF and does in general contribute to a
or IDH2. Myelofibrotic progression consistent with post-
worsening of prognosis [50].
PV MF is reported to range between 6 and 14% at 15 years
Diagnostic criteria for the diagnosis of PV according to
[45–47].
the ICC are reported in Table 4.

Morphology
Myeloproliferative neoplasm, unclassifiable
The diagnostic thresholds for hemoglobin/hematocrit have
not been changed by the ICC and therefore an acquired Clinical features
increase in hemoglobin/hematocrit level above 16.5 gm/
dL/49% in men and 16 g/dL/48% in women, in the con- MPN-U share clinical, morphological, and molecular
text of a JAK2 mutation and characteristic bone marrow features of MPN but do not fulfill the diagnostic criteria
morphology define this MPN subtype. The peripheral of a specific subtype. They account for about 5–10% of
blood shows a mild to overt excess of normochromic, all MPN cases and can be subdivided in (i) early phase
normocytic RBCs. Neutrophilia and rarely basophilia MPN; (ii) advanced fibrotic phase MPN; and (iii) MPN
may be present. The bone marrow is in almost all cases with concurrent inflammatory or neoplastic disorders
hypercellular for the patient’s age due to the proliferation obscuring the underlying MPN. The clinical presentation
of all three cell lineages (so-called panmyelosis). Eryth- of MPN-U is variable: early phase MPN-U may display
ropoiesis and granulopoiesis frequently show a left-shift, increased blood cell counts (thrombocytosis and/or leuko-
and the myeloid/erythroid ration can be variable (Fig. 3). cytosis and/or erythrocytosis) usually without significant
Megakaryocytes are generally increased in number and splenomegaly or hepatomegaly, while advanced stages are
are characterized by a marked polymorphism (marked commonly characterized by cytopenia, anemia, and orga-
variation in size) without any significant atypia [27, 48]. nomegaly. Along these lines, about 50% of MPN patients
Loose clusters of megakaryocytes are a common feature presenting with splanchnic vein thrombosis reveal over-
in polycythemic stages of disease, whereas atypical dense lapping clinical and morphological features and thus are
and/or huge clusters as described in PMF might occur in often classified as MPN-U [53].

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Virchows Archiv (2023) 482:53–68 65

Morphology Bleeding diathesis, including a high incidence of cerebral

hemorrhage, can be also related to CNL [57].
In early phase MPN-U, morphological features of a specific
MPN subtype are not fully developed, and many cases show Morphology
overlapping features between ET and pre-PMF. Noteworthy,
the reduction of the required hemoglobin/hematocrit thresh- Bone marrow is usually markedly hypercellular (> 90%
olds for the diagnosis of PV by the previous WHO criteria cellularity) due to the proliferating granulopoiesis with
has significantly reduced the number of unclear cases [54, a prevalence of metamyelocytes and segmented granu-
55]. If the initial diagnosis of MPN is established in the overt locytes leading to an increased myeloid/erythroid ratio
fibrotic phase displaying advanced stromal alterations (col- which may exceed 20:1 (Fig. 4). Erythropoiesis is usually
lagen deposition, increased micro-vessel density, sinusoid normal, while megakaryocytes may be slightly increased,
ectasia, and bone remodeling), demonstration of a character- but with normal morphology. Myeloblasts usually account
istic driver mutation is important to establish the diagnosis, for less than 5% of the bone marrow cells. Monocytosis,
however, without assignment to a specific subtype like PMF basophilia, eosinophilia, or significant dysgranulopoiesis
and post-ET or post-PV MF. are usually absent, and their presence should prompt a
critical review of diagnosis in order to separate the case
Genetic profile from MDS/MPN overlaps. Mild increase in reticulin fibro-
sis (MF-1) can be seen in a minority of cases. In line with
Diagnosis of MPN-U can be challenging and requires the the other MPN subtypes, transformation to AP is defined
exclusion of reactive conditions, such as infections and toxin by 10 to 19% of peripheral blood or bone marrow blasts
or drug exposure (growth factors, cytokines, or immunosup- and frequently associated with progressive splenomegaly
pressive drugs). In this context, documentation of clonality and worsening of thrombocytopenia. Accordingly, ≥ 20%
of hematopoiesis by identification of MPN driver muta- blasts define BP. In cases presenting with a CSF3RT618I
tions, or other mutations associated with myeloid neoplasms or other activating CSF3R mutation, the ICC guidelines
(ASXL1, EZH2, TET2, IDH1/ IDH2, SRSF2, and SF3B1), propose to lower the key diagnostic threshold for leuko-
support the diagnosis [56]. About 20–30% of patients reveal cytosis from ≥ 25 to ≥ 13 × ­1 0 9/L [58–60]. Because a
cytogenetic abnormalities, which also support the diagno- marked neutrophilic increase can accompany different
sis. Marked dysplastic changes and a lack of MPN driver benign and malignant disorders, proper integration of
mutations should prompt a careful diagnostic workup to clinical and morphological findings is mandatory for the
separate these cases from MDS/MPN overlaps. In addition, correct differential diagnosis, in particular in molecular
it is important to highlight that a diagnosis of MPN-U can- undefined cases. In context, the differential diagnosis
not be made in cases with genetic lesions defining specific includes reactive neutrophilia/leukemoid reaction, CML,
myeloid neoplasms (BCR::ABL1 fusion, myeloid/lymphoid and myelodysplastic/myeloproliferative neoplasms such as
neoplasms with eosinophilia and gene rearrangement). atypical chronic myeloid leukemia (aCML) and chronic
Diagnostic criteria for the diagnosis of CNL according to myelomonocytic leukemia (CMML), as well as other
the ICC are reported in Table 5. myeloid neoplasms.

Genetic profile
Chronic neutrophilic leukemia
The presence of a driver mutation in the colony stimulat-
Clinical features ing factor 3 receptor (CSF3R) is the defining genetic sig-
nature of CNL. It can be identified in 80–100% of cases,
CNL is a rare BCR::ABL1-negative MPN subtype with but the absence of a CSF3R mutation does not exclude the
an overall incidence of 0.1 cases/1,000,000 presenting possibility of CNL. Among the CSF3R-mutated patients,
in patients with a median age at diagnosis of 66.5 years two molecular subgroups (T618I vs other CSF3R mutations)
(range: 15–86) and neutrophilic leukocytosis. In most with phenotypic and prognostic differences have been identi-
patients, leukocytosis precedes the diagnosis for several fied [61]. The CSF3RT618I-mutated subset clustered with
months. Rarely patients present with symptoms, such as adverse clinical and laboratory features, more advanced age
fatigue, bone pain, pruritus, easy bruising, or gout. Sple- at diagnosis, higher white blood cell counts, lower hemo-
nomegaly (and hepatomegaly) of various degree is a fre- globin values and platelet counts at diagnosis, more fre-
quent finding and palpable splenomegaly can be detected quently abnormal karyotype, and a lower overall survival
in about 36% of CSF3R-mutated cases at diagnosis. in comparison to cases harboring other CSF3R mutations.

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66 Virchows Archiv (2023) 482:53–68

As in other MPN subtypes, additional prognostic relevant Declarations

mutations can be seen in many cases including SETBP1,
ASXL1, and SRSF2. Ethics approval Not applicable.
Diagnostic criteria for the diagnosis of CEL, NOS accord-
ing to the ICC are reported in Table 6. Conflict of interest The authors declare no competing interests.

Open Access This article is licensed under a Creative Commons Attri-

bution 4.0 International License, which permits use, sharing, adapta-
Chronic eosinophilic leukemia, tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
not otherwise specified provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
CEL, NOS is characterized by persistent eosinophilia not included in the article's Creative Commons licence, unless indicated
meeting the criteria for other genetically defined entities. otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
Mutations detected by NGS help to establish clonality in
permitted by statutory regulation or exceeds the permitted use, you will
a significant subset of cases with eosinophilic disorders need to obtain permission directly from the copyright holder. To view a
[62–64]. The bone marrow in CEL typically is hypercellu- copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/.
lar and reveals dysplastic megakaryocytes, with or without
dysplastic features in other lineages, and often a significant
fibrosis which is associated with the eosinophilic infiltrate. References
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