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An update on BCR-ABL1-negative myeloproliferative neoplasms
MINI-SYMPOSIUM: BONE MARROW PATHOLOGY
An update on BCR-ABL1negative myeloproliferative neoplasms
PDGFRA, PDGFRB or FGFR1, will not be considered here as the diagnostic process is different. Myeloproliferative neoplasms are clonal haemopoietic stem cell disorders, characterized by proliferation of one or more of the myeloid cell lineages and lack of significant dysplasia. Classification continues to be influenced by recent discoveries of genetic abnormalities involved in the pathogenesis of BCR-ABL1negative MPNs, correlated with specific histological features and clinical findings.
Jon D van der Walt
Abstract BCR-ABL 1 negative myeloproliferative neoplasms (MPN) are clonal haemopoietic stem cell disorders, characterized by proliferation of one or more of the myeloid cell lineages. Classification is based on the genetic abnormalities involved in the pathogenesis and on correlation of specific histological features with clinical findings. The diagnostic approach must take into account clinical, haematological, morphological and genetic features. A clinico-pathologically assigned diagnosis therefore forms the basis of the current consensus based classification. Myeloproliferative neoplasms may have an insidious onset and variable progression, comprising the full spectrum of evolution from a prodromal or initial phase, through a stable phase, to a terminal phase. The latter results in bone marrow failure, myelodysplastic changes and blast crisis. Prodromal stages pose a number of diagnostic problems because they may not present with the classic clinico-pathological criteria for diagnosis. Follow-up biopsies are important to monitor progression and features of regression, stability, progression and the effects of therapy must be noted. In the MPN group particularly, quantitation of fibrosis is very important and it must be graded accurately throughout the course of the disease. The effect of therapies must be taken into account in initial and follow-up biopsies. The last decade has seen tremendous advances in molecular biology, diagnosis and treatment of BCR-ABL1-neg MPN. These advances have led to re-examination of diagnostic criteria and revision of the 2008 WHO classification is required urgently.
Approach to diagnosis The diagnostic approach must take into account clinical, haematological, morphological and genetic features (Box 1). A clinico-pathologically assigned diagnosis forms the basis of the current consensus classification (Box 2). This classification is not without controversy and alternative views have been expressed.2 Reproducibility of the histopathology of certain aspects of the classification also remains controversial. Myeloproliferative neoplasms may have an insidious onset and variable progression, comprising the full spectrum of evolution from a prodromal or initial phase, through a stable phase, to a terminal phase. The latter results in bone marrow failure, myelodysplastic changes and blast crisis (Table 1 ). Prodromal stages pose a number of diagnostic problems because they may not present with the classic clinicopathological criteria for diagnosis.3,4 They may be recognisable by bone marrow examinations but may also result in an “unclassifiable” diagnosis. Rebiopsy may eventually clarify the situation but these cases may initially have to be placed in the MPN, U or MDS/MPN, U category awaiting follow-up investigations and eventual reclassification. Likewise, the onset of ‘acceleration’ and acute transformation is sometimes difficult to define and there is a lack of consensus on diagnostic criteria. The WHO criteria of 20% blasts in blast crisis and 10e19% blasts in accelerated phase may be difficult to apply in practice in cases of ‘dry tap’ marrow aspiration and discordant bone marrow and peripheral blood findings. It is in any case arbitrary. A blast crisis may be the result of clonal evolution that is obvious from an increasingly complex karyotype. During the phase of progression, transition to an MDS/MPN phenotype preceding transformation is often parallelled by development of a complex karyotype typical for secondary AML.5 It should also be noted that extramedullary blast proliferation indicates blast phase and that the marrow findings may be discordant. Follow-up biopsies are important to monitor progression and features of regression, stability, progression and the effects of therapy must be noted (Table 2 ). In the MPN group particularly, quantitation of fibrosis is very important and it must be accurately graded (Box 3). The effects of therapies such as hydroxycarbamide must be taken into account. The differential diagnosis in MPN includes other members of this category, the related refractory anaemia with ring sideroblasts with thrombocytosis (RARS-T)6 and also entities such as MPN/MDS and MDS with isolated del(5q), which may present with thrombocytosis.7 A variety of reactive disorders must be actively excluded before a diagnosis of MPN or MDS/MPN can be established. Of particular interest are the hereditary
Keywords chronic eosinophilic leukaemia; chronic neutrophilic leukaemia; essential thrombocythaemia; myeloproliferative neoplasm; polycythaemia vera; primary myelofibrosis
Introduction In the fourth edition of the WHO classification,1 the term ‘chronic myeloproliferative disease’ was replaced by ‘myeloproliferative neoplasm’ (MPN). Inclusion of systemic mastocytosis (SM) has attracted controversy and, together with the myeloid and lymphoid neoplasms with eosinophilia and abnormalities of
Abbreviations: MPN, myeloproliferative neoplasm; MPN, U, myeloproliferative neoplasm, unclassifiable; MDS, myelodysplastic syndromes; MPN/MDS, myeloproliferative neoplasm/myelodysplastic syndrome; PV, polycythaemia vera; PMF, primary myelofibrosis; ET, essential thrombocythaemia; CML, chronic myelogenous leukaemia; CNL, chronic neutrophilic leukaemia; CEL, chronic eosinophilic leukaemia; AML, acute myeloid leukaemia; CMML, chronic myelomonocytic leukaemia; SM, systemic mastocytosis. Jon D van der Walt MB BCH FRCPATH, Consultant Histopathologist Department of Histopathology, Guy’s and St Thomas’ Hospitals, London, UK. Conflicts of interest: none declared.
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myeloproliferative disorders,8 hereditary thombocytosis9 and congenital erythrocytosis.10,11 Knowledge of the family history and appreciation of variant morphology are crucial for the diagnosis. With the decline in biopsy rates for PV in the post JAK2V617F era, a higher proportion of cases of congenital or acquired erythrocytosis are likely to encountered. Reactive conditions such as infection with HIV12 may cause thrombocytosis. The use of thrombopoietin receptor agonists in thrombocytopenic states13 may mimic ET or PV, with increased reticulin and MPN-like morphology.
Guidelines for using the revised WHO classification of myeloid neoplasms26 Specimen requirements C PB and BM specimens collected prior to any definitive therapy. C PB and cellular BM aspirate smears and/or touch preparations stained with Wright-Giemsa or similar stain. C BM biopsy specimen, at least 1.5 cm in length and at right angles to the cortical bone, is recommended for all cases if feasible. C BM specimens for complete cytogenetic analysis and, when indicated, for flow cytometry, with an additional specimen cryopreserved for molecular genetic studies. The latter studies should be performed based on initial karyotypic, clinical, morphologic, and immunophenotypic findings.
Advances in the molecular pathology of MPN Acquired somatic mutations of JAK2V617F and JAK2 exon 12 have been shown to play a crucial role in the pathogenesis of many cases of BCR-ABL1-negative MPN and these discoveries opened a new era in the understanding of the biology and therapy of MPN.14,15 JAK2V617F mutation is detected in about 95% of patients with PV as a secondary genetic event that is preceded by an as yet undefined molecular abnormality. As a consequence of this mutation, transformation and proliferation of haematopoietic progenitor cells are promoted by downstream signal transduction pathways. In ET and PMF, this mutation is found in only about 50% of patients and in JAK2V617F negative PV, an activating JAK2 exon 12 mutation is detectable.16 In a small number of patients with ET and PMF, an activating mutation of MPLW515L/K is present.17 It must be noted that JAK 2V617F is not specific for MPN and its absence does not exclude the diagnosis. In 2013, two groups independently reported novel calreticulin gene (CALR) mutations (exon 9 deletions and insertions) in JAK2 or MPL unmutated PMF and ET.18,19 In one of the studies, among 1107 patient samples with MPN, CALR mutations were not seen in PV but were seen in ET (25%) and PMF (35%).19 CALR mutations were mutually exclusive of JAK2 or MPL mutations. When JAK2/MPL-unmutated cases were analysed, CALR mutational were found to be 67% in ET and 88% in PMF. CALR mutations were not detected in AML, MDS, CMML or CML. Three patients (13%) with RARS-T displayed CALR mutations and all three were JAK2/MPL-negative. Subsequent studies of CML cases have shown concurrent CALR and BCR-ABL1; these have been described20,21 as ‘ET evolving to CML’ and ‘A CALR Mutation Preceding BCR-ABL1 in an Atypical Myeloproliferative Neoplasm’. These complex cases require synthesis of haematological, morphological and molecular findings to reach a diagnosis. Other mutations, including TET2, IDH, ASXL1 or DNMT3A, are occasionally seen in PV and ET.22 The presence or absence of the various mutations has been found to have prognostic implications. In a recent study of PMF, CALR mutations were mutually exclusive of JAK2 or MPL mutations and were associated with younger age and higher platelet counts, with patients being less likely to be anaemic, require transfusions or display leucocytosis. CALR mutations had a favourable impact on survival while ‘CALR-ASXL1þ’ and ‘triple-negative’ cases were identified as high-risk molecular signatures in PMF.23 In another study, ET patients with CALR mutation had lower leucocyte counts and ages compared with JAK2-mutated ET patients.24 Thus, evaluation of JAK2, MPL, and CALR mutation status has become mandatory not only for diagnosis but also for prognostication.25
Assessment of blasts C Blast percentage in PB and BM is determined by visual inspection. C Myeloblasts, monoblasts, promonocytes, megakaryoblasts (but not dysplastic megakaryocytes) are counted as blasts when summing blast percentage for diagnosis of AML or blast transformation; count abnormal promyelocytes as “blast equivalents” in APL. C Proerythroblasts are not counted as blasts except in rare instances of “pure” acute erythroleukaemia. C Flow cytometric assessment of CD34þ cells is not recommended as a substitute for visual inspection; not all blasts express CD34, and artifacts introduced by specimen processing may result in erroneous estimates. C If the aspirate is poor and/or marrow fibrosis is present, immunohistochemistry (IHC) on biopsy sections for CD34 may be informative if blasts are CD34þ. Assessment of blast lineage C Multiparameter flow cytometry (at least three colours) is recommended; the panel should be sufficient to determine lineage as well as aberrant antigen profile of a neoplastic population. C Cytochemistry, such as myeloperoxidase or nonspecific esterase, may be helpful, particularly in AML, NOS, but it is not essential in all cases. C IHC on biopsy sections may be helpful; many antibodies are now available for recognition of myeloid and lymphoid antigens. Assessment of genetic features C Complete cytogenetic analysis from BM at initial diagnosis when possible. C Additional studies, such as FISH, RT-PCR, mutational status, should be guided by clinical, laboratory, and morphologic information. C Mutational studies for mutated NPM1, CEBPA, and FLT3 are recommended in all cytogenetically normal AML; mutated JAK2 should be sought in BCR-ABL1enegative MPN, and mutational analysis for MPL, CALR, KIT, NRAS, PTNP11, etc, should be performed as clinically indicated. Correlation/reporting of data C All data should be assimilated into one report that states the WHO diagnosis. Box 1
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WHO 2008 characterisation of BCR-ABL1 negative MPN1
Minor criteria C Leukoerythroblastosis C Increase in serum lactate dehydrogenase level C Anaemia C Palpable splenomegaly
Polycythaemia Vera (PV) Figure 1aed . Diagnosis requires the presence of both major criteria and one minor criterion or the presence of the first major criterion and two minor criteria. Major criteria C Hgb >18.5 g/100 ml in men or 16.5 g/100 ml in women or other evidence of increased red cell volume. (Hgb or Hct >99th percentile of reference range for age/sex/altitude or Hgb >99th percentile of reference range for age/sex/altitude or Hgb >17 g/ 100 ml in men or 15 g/100 ml in women if associated with a documented and sustained increase of at least 2 g/100 ml from an individual’s baseline value that is not attributed to correction of iron deficiency anaemia, or elevated red cell mass (>25% above mean normal predicted value.). C Presence of the JAK 2V617F or other functionally similar mutations, such as the JAK2 exon 12 mutation. Minor criteria C BM showing hypercellularity for age with trilineage growth (panmyelosis) and with prominent erythroid and megakaryocytic proliferation. C Serum Epo level below the reference range for normal. C Endogenous erythroid colony formation in vitro
Chronic neutrophilic leukaemia (CNL) C Peripheral blood leucocytosis 25 109/Litre C Hypercellular bone marrow biopsy C Mature neutrophils C No dysplasia C Hepatosplenomegaly C Clonality established or reactive causes excluded C No BCR-ABL1 fusion gene C No rearrangement of PDGFRA, PDGFRB or FGFR1 C No evidence of PV, PMG or ET C No evidence of MDS or MDS/MPN C Slowly progressive
Chronic eosinophilic leukaemia (CEL) C Eosinophilia (1.5 109/Litre) C No Ph chromosome, no BCR-ABL1 fusion gene C No evidence of other MPN, MDS/MPN, AML C No rearrangement of PDGFRA, PDGFRB or FGFR1 C Clonal cytogenetic or molecular genetic abnormality or blasts >2% in PB or >5% in BM. C Blast count <20% C Immunophenotypically aberrant T-lymphocytosis is excluded
Essential thrombocythaemia (ET) Figure 2a . Diagnosis requires meeting all 4 criteria. C C
C
C
Sustained platelet count 450 109/Litre* Bone marrow biopsy specimen showing proliferation mainly of the megakaryocytic lineage with increased numbers of enlarged, mature megakaryocytes. No significant increase or left-shift of neutrophil granulopoiesis or erythropoiesis. Not meeting WHO criteria for polycythemia vera, primary myelofibrosis, BCR-ABL1epositive CML, or MDS or other myeloid neoplasm. Demonstration of JAK 2V617F or other clonal marker, or in the absence of JAK 2V617F, no evidence of reactive thrombocytosis.
Myeloproliferative neoplasm, unclassifiable (MPN, U) C Definite clinical, laboratory or morphological features of MPN C Does not meet criteria for specific MPN or has overlapping features C No BCR-ABL1 fusion gene C No rearrangement of PDGFRA, PDGFRB or FGFR1 C Not used for inadequately investigated cases C Reactive or post therapeutic changes must be excluded C Prodromal or late phase MPN cases are most common
Primary myelofibrosis (PMF) Figure 2bed. Diagnosis requires meeting all 3 major criteria and 2 minor criteria. Major criteria C Presence of megakaryocyte proliferation and atypia,* usually accompanied by either reticulin or collagen fibrosis, or, in the absence of significant reticulin fibrosis, the megakaryocyte changes must be accompanied by an increased bone marrow cellularity characterized by granulocytic proliferation and often decreased erythropoiesis (ie, prefibrotic cellular-phase disease). C Not meeting WHO criteria for PV, CML, MDS, or other myeloid disorders. C Demonstration of JAK 2V617F or other clonal marker (eg, MPLW515K/ L ), or, in the absence of the above clonal markers, no evidence that bone marrow fibrosis is secondary to infection, autoimmune disorder or other chronic inflammatory condition, hairy cell leukemia or other lymphoid neoplasm, metastatic malignancy, or toxic (chronic) myelopathies.
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Box 2
Prodromal states Controversy has long surrounded the diagnosis of cases of MPN that, at presentation, do not manifest all of the classical diagnostic findings but which may subsequently progress. While the diagnosis of overt PV is usually straightforward, particularly since the establishment of JAK2617VF or other functionally similar mutation such as JAK2 exon 12 mutation as a major diagnostic criterion, prodromal-latent stage or socalled ‘masked’ PV may be difficult to assess. Recent proposals26 have called for reduction in the defining haemoglobin levels in the WHO classification, or the introduction of haematocrit as a diagnostic criterion, as proposed by the British Committee for Standards in Haematology.27 The value of bone marrow biopsy is emphasized in these difficult cases of MPN26
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Staging of myelofibrosis (MF) in MPN33
Diagnosis: The three phases of MPN Evolution
Stability
Progression
Criteria incomplete Diagnostic confusion
Classic morphology
Transitional criteria
Criteria well defined
Morphological changes Cytogenetic changes
Cytogenetics stable Diagnostic problems Reactive vs MPN Variant morphology & cytogenetics MPN, U
MF 1 (Reticulin, focal, patchy, endophytic bone formation absent) C Loose network of reticulin with intersections, especially in peripheral areas, no collagenisation. C Sinusoidal Haematopoiesis is absent.
MF 2 (Reticulin plus collagen, diffuse meshwork, endophytic bone formation may be present) C Diffuse increase in reticulin with extensive intersections and only focal bundles of collagen. Hypercellular bone marrow. C Sinusoidal haematopoiesis may be present.
Accelerated vs acute phase Quantitation of fibrosis
MF3 (Mostly collagen, diffuse meshwork, scarring, dilated sinuses, endophytic bone formation scant to prominent) C Diffuse and dense increased reticulin with extensive intersections with course bundles of collagen and significant osteosclerosis. C Sinusoidal haematopoiesis may be prominent.
ET vs pf-PMF PV vs MPN, U Table 1
and also in the distinction from cases of erythrocytosis, in which the classical morphology of MPN is lacking.10,11 It has been noted that the presence of a CALR mutation excludes a diagnosis of PV.28 Some authors have stressed the importance of so called prefibrotic PMF, which can mimic ET in its presentation. The mutation profile is similar for JAK2, CALR and MPL mutations according to these authors.29 It is emphasized that careful morphologic examination of bone marrow histology is necessary to distinguish the two; megakaryocytes in ET being large and mature-appearing whereas those in prefibrotic PMF display abnormal maturation with hyperchromatic and irregularly folded nuclei.4 The distinction between ET and prefibrotic PMF is claimed to be prognostically relevant.30 However, other authors have disputed the reproducibility of the diagnosis.31,32 Clarification is expected in the revised WHO classification.
Box 3
criteria34 in order to establish the point in the progression of MPN at which myelofibrosis develops. It should be noted that it is essential to document a previous diagnosis of ET or PV before confirming a diagnosis of post-PV or post-ET fibrosis. Distinction from PMF may be impossible if a well-documented preceding diagnosis, preferably with biopsy confirmation, is not available (Box 4). Risk stratification according to mutation status and other factors, and management options, have recently been reviewed.29,35 A recent large study36 has shown that the newly discovered mutations have yet to render superfluous the need for morphologic distinction of ET from PV both diagnostically and prognostically and that determination of JAK2/CALR mutational status alone, without morphologic examination, did not differentiate PV from JAK2 mutant ET, while distinguishing morphologically defined ET into JAK2 and CALR subtypes carried limited prognostic relevance. It has recently been recognized that the development of absolute monocytosis in patients with established PMF is associated with rapid disease progression and these patients have a shorter survival.37
Fibrosis and progression All MPN are prone to develop fibrosis and accurate assessment of grade of reticulin fibrosis33 is fundamental to the assessment of bone marrow histology in these patients, at time of diagnosis and on follow-up biopsies. It is necessary to adhere to strictly defined
Chronic neutrophilic leukaemia
Diagnosis: Assessment of follow-up biopsy samples in MPN and MDS/MPN Regression
Stability
Progression
Therapy
Reticulin reduced
Diagnosis unchanged or clarified Reticulin & cellularity stable
Reticulin increased
Normalisation
Spent phase
Cellularity reduced
Acute phase
Dysplasia Acute phase
Cellularity reduced
The current WHO diagnostic criteria for CNL (Table 1) were confounded by the absence of a clonal marker and defining bone marrow morphology. A relatively high leucocyte count threshold was required and reactive granulocytosis, plasma cell neoplasmassociated neutrophilia, CML, aCML and CMML thus had to be excluded. Recently, recurrent somatic mutations in the proximal domain of the gene for colony-stimulating factor 3 receptor (CSF3R) were reported in patients with CNL.38,39 CSF3R was mutated in 100%, SETBP1 in 33%38 and CALR in 12.5% of WHOdefined cases of CNL.40 The original publications38 suggested that somatic CSF3RT618I mutations were present in CNL and aCML. A subsequent
Table 2
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Figure 1 a) PV showing classical panmyelosis and polymorphic megakaryocytes. b) PV with JAK2 exon 12 mutation showing lack of megakaryocyte clustering. c). PV showing progression to grade 2 myelofibrosis. d). PV showing progression to acute myeloid leukaemia.
Figure 2 a). Classical ET showing large mature megakaryocytes with ‘staghorn nuclei’. b). Cellular phase of PMF showing hyperchromatic, abnormal folded nuclei. c). Grade 3 osteomyelofibrosis showing typical megakaryocyte morphology. d). Post hydroxycarbamide changes. A morphological diagnosis of MPN type in not possible.
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manifestations mimicking CNL.45 However, cases of proven CNL with concurrent plasma cell neoplasia have also been described.46 The mechanism by which CNL occurs in association with a plasma cell neoplasm remains unknown and a coincidence is possible.47 Cases of patients with CNL receiving therapy with the JAK1/2 inhibitor ruxolitinib and showing a clinical response have been reported, opening the door to a new therapeutic approach.38 Progression in CNL is not as well defined as in the more common MPN categories but therapy-resistant progressive neutrophilia, thrombocytopenia, splenomegaly, clonal evolution and myeloid blast crisis are recognized.43 Revision of the current WHO classification is expected to include the molecular criterion of mutated CSF3R in the diagnostic criteria for CNL.43,45 A recent proposal principally includes peripheral blood leucocytosis 13 109/litre, >80% neutrophils or bands and the presence of CSF3RT618I or other membrane proximal CSF3R mutations and lack of dysgranulopoiesis. There must be no monocytosis, molecular evidence of BCR-ABL-1, PDGFRA, PDGFRB, or FGRF1 rearrangement.48
Recommended criteria for post-polycythemia vera and post-essential thrombocythemia myelofibrosis34 Criteria for post-polycythemia vera myelofibrosis Required criteria: C
C
Documentation of a previous diagnosis of polycythemia vera as defined by the WHO criteria Bone marrow fibrosis grade 2e3 (on 0e3 scale) or grade 3e4 (on 0e4 scale)
Additional criteria (two are required): C
C C
C
Anaemia or sustained loss of requirement for phlebotomy in the absence of cytoreductive therapy A leucoerythroblastic peripheral blood picture Increasing splenomegaly defined as either an increase in palpable splenomegaly of 5 cm (distance of the tip of the spleen from the left costal margin) or the appearance of a newly palpable splenomegaly Development of 1 of three constitutional symptoms: >10% weight loss in 6 months, night sweats, unexplained fever (>37.5 C)
Chronic eosinophilic leukaemia In contrast to CNL, CEL still remains a diagnosis of exclusion. Secondary eosinophilia has numerous causes and infections, allergic reactions, autoimmune diseases, lymphoma and rare congenital conditions have to be investigated by specialists in different disciplines.49 Once reactive causes are excluded, morphological examination of the bone marrow will be required to consider eosinophilia associated with other WHO entities such as SM, CML, MDS, AML, CMML etc49 and the lymphoid variant of hypereosinophilic syndrome.50 This is defined by the presence of a Th2 T-cell subset overproducing interleukin-5. Many different abnormal T-cell immunophenotypes have been described, the most frequent of which are: CD3 CD4þ, CD3þ CD4þ CD7, and CD3 þ CD4 CD8 TCRabþ. Clonal rearrangement of TCR genes is frequently observed in these abnormal T-cells.50 Investigations should be directed to exclude Philadelphia chromosome or BCR-ABL1 fusion, t(5;12)(q31-q35;p13) or other rearrangement of PDGFRB, FIP1L1-PDGFRA fusion or other rearrangement of PDGFRA and no rearrangement of FGFR1, inv(16)(p13q22) or t(16;16)(p13;q22), or other features diagnostic of AML must be present. Clonal cytogenetics or other abnormal cytogenetics must be established in addition to these exclusions.50
Criteria for post-essential thrombocythemia myelofibrosis Required criteria: C
C
Documentation of a previous diagnosis of essential thrombocythemia as defined by the WHO criteria (see Table 2) Bone marrow fibrosis grade 2e3 (on 0e3 scale) or grade 3e4 (on 0e4 scale)
Additional criteria (two are required): C
C C
C C
Anaemia and a 2 g/dL decrease from baseline haemoglobin level A leucoerythroblastic peripheral blood picture Increasing splenomegaly defined as either an increase in palpable splenomegaly of 5 cm (distance of the tip of the spleen from the left costal margin) or the appearance of a newly palpable splenomegaly Increased lactate dehydrogenase Development of of three constitutional symptoms: >10% weight loss in 6 months, night sweats, unexplained fever (>37.537.5 C)
Box 4
Conclusion publication denied the association with aCML41 but other authors have found it in small number (3%) of patients with aCML.42 The JAK2V617F mutation has also been reported in CNL but its significance is unclear.43 Neutrophilic progression in a case of PV mimicking CNL has been described, emphasizing the need for careful work-up in complex cases.44 CSF3R mutations have also been reported in hereditary chronic neutrophilia.43 There is a well established association between neutrophilia and plasma cell neoplasia. Granulocyte-colony stimulating factor-producing myeloma has been described, with clinical
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The last decade has seen tremendous advances in molecular biology, diagnosis and treatment of BCR-ABL1-negative MPN. These advances have led to re-examination of diagnostic criteria and revision of the 2008 WHO classification is required urgently, which is expected in 2016. A REFERENCES 1 Swerdlow SH, Campo E, Harris NL, et al. WHO classification of tumours of haematopoietic and lymphatic tissues. Lyon: IARC, 2008.
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23 Tefferi A, Lasho TL, Finke CM, et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia 2014; 28: 1472e7. 24 Ha J-S, Kim Y-K. Calreticulin exon 9 mutations in myeloproliferative neoplasms. Ann Lab Med 2015; 35: 22e7. 25 Cazzola M, Kralovics R. From Janus kinase 2 to calreticulin: the clinically relevant genomic landscape of myeloproliferative neoplasms. Blood 2014; 123: 3714e9. 26 Barbui T, Thiele J, Vannucchi AM, et al. Rethinking the diagnostic criteria of polycythemia vera. Leukemia 2014; 28: 1191e5. 27 McMullin MF, Bareford D, Campbell P, et al. Guidelines for the diagnosis, investigation and management of polycythaemia/erythrocytosis. Br J Haematol 2005; 130: 174e95. 28 Barbui T, Thiele J, Carobbio A, et al. Discriminating between essential thrombocythemia and masked polycythemia vera in JAK2 mutated patients. Am J Hematol 2014; 89: 588e90. 29 Tefferi A, Pardanani A. Genetics: CALR mutations and a new diagnostic algorithm for MPN. Nat Rev Clin Oncol 2014; 11: 125e6. 30 Barbui T, Thiele J, Passamonti F, et al. Survival and disease progression in essential thrombocythemia are significantly influenced by accurate morphologic diagnosis: an international study. J Clin Oncol 2011; 29: 3179e84. 31 Brousseau M, Parot-Schinkel E, Moles M-P, et al. Practical application and clinical impact of the WHO histopathological criteria on bone marrow biopsy for the diagnosis of essential thrombocythemia versus prefibrotic primary myelofibrosis. Histopathology 2010; 56: 758e67. 32 Buhr T, Hebeda K, Kaloutsi K, et al. European Bone Marrow Working Group trial on reproducibility of World Health Organization criteria to discriminate essential thrombocythemia from prefibrotic primary myelofibrosis. Haematologica 2012; 97: 360e5. 33 Thiele J, Kvasnicka HM, Facchetti F, et al. European consensus on grading bone marrow fibrosis and assessment of cellularity. Haematologica 2005; 90: 1128e32. 34 Barosi G, Mesa RA, Thiele J, et al. Proposed criteria for the diagnosis of post-polycythemia vera and post-essential thrombocythemia myelofibrosis: a consensus statement from the International Working Group for Myelofibrosis Research and Treatment. Leukemia 2008; 22: 437e8. 35 Tefferi A, Barbui T. Polycythemia vera and essential thrombocythemia: 2015 update on diagnosis, risk-stratification and management. Am J Hematol 2015; 90: 163e73. 36 Tefferi A, Guglielmelli P, Larson DR, et al. Long-term survival and blast transformation in molecularly annotated essential thrombocythemia, polycythemia vera, and myelofibrosis. Blood 2014; 124: 2507e13. 37 Boiocchi L, Espinal-Witter R, Turbiner Julia, et al. Development of monocytosis in patients with primary myelofibrosis indicates an accelerated phase of the disease. Mod Pathol 2013; 26: 204e12. 38 Maxson JE, Gotlib J, Pollyea DA, et al. Oncogenic csf3r mutations in chronic neutrophilic leukemia and atypical CML. N Engl J Med 2013; 368: 1781e90. 39 Pardanani A, Lasho TL, Laborde RR, et al. CSF3R T618I is a highly prevalent and specific mutation in chronic neutrophilic leukemia. Leukemia 2013; 27: 1870e3. 40 Lasho TL, Elliott MA, Pardanani A, et al. CALR mutation studies in chronic neutrophilic leukemia. Am J Hematol 2014; 89: 450.
2 Spivak JL, Silver RT. The revised World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia and primary myelofibrosis: an alternative proposal. Blood 2008; 112: 231e9. 3 Kvasnicka HM, Thiele J. Diagnosis of chronic myeloproliferative neoplasms with special emphasis on early stages. Diag Histopath 2009; 15: 125e33. 4 Kvasnicka HM, Thiele J. Prodromal myeloproliferative neoplasms: the 2008 WHO classification. Am J Hematol 2010; 85: 62e9. €hring G, Schlegelberger B. Progression of 5 Kreipe H, Hussein K, Go myeloproliferative neoplasms to myelofibrosis and acute leukaemia. J Hematopathol 2011; 4: 61e8. 6 Patnaik MM, Tefferi A. Refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). Am J Hematol 2015 Apr 21 [Epub ahead of print]. 7 Patnaik MM, Lasho TL, Finke CM, et al. Isolated del(5q) in myeloid malignancies: clinicopathologic and molecular features in 143 consecutive patients. Am J Hematol 2011; 86: 393e8. 8 Skoda RC. Hereditary myeloproliferative disorders. Haematologica 2010; 95: 6e8. 9 Skoda RC. Thrombocytosis. Hematology Am Soc Hematol Educ Program 2009; 1: 159e67. 10 McMullin MF. The classification and diagnosis of erythrocytosis. Int Jnl Lab Hem 2008; 30: 447e59. 11 Patnaik MM, Tefferi A. The complete evaluation of erythrocytosis: congenital and acquired. Leukemia 2009; 23: 834e44. 12 Miguez-Burbano MJ, Burbano X, Rodriguez A, et al. Development of thrombocytosis in HIVþ drug users: impact of antiretroviral therapy. Platelets 2002; 13: 183e5. 13 Brynes RK, Orazi A, Theodore D, et al. Evaluation of bone marrow reticulin in patients with chronic immune thrombocytopenia treated with eltrombopag: data from the EXTEND study. Am J Hematol 2015 Mar 19 [Epub ahead of print]. 14 Kralovics R, Passamonti F, Buser AS, et al. A gain-of function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005; 352: 1779e90. 15 Jones AV, Kreil S, Zoi K, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood 2005; 106: 2162e8. 16 Scott LM. The JAK2 exon 12 mutations: a comprehensive review. Am J Hematol 2011; 86: 668e76. 17 Tefferi A. JAK and MPL mutations in myeloid malignancies. Leuk Lymphoma 2008; 49: 388e97. 18 Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. New Engl J Med 2013; 369: 2391e405. 19 Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. New Engl J Med 2013; 369: 2379e90. 20 Bonzheim I, Mankel B, Klapthor P, et al. CALR-mutated essential thrombocythemia evolving to chronic myeloid leukemia with coexistent CALR mutation and BCR-ABL translocation. Blood 2015; 125: 2309e11. 21 Cabagnols X, Cayuela JM, Vainchenker W. A CALR mutation preceding BCR-ABL1 in an atypical myeloproliferative neoplasm. N Engl J Med 2015; 372: 688e90. 22 Tefferi A. Primary myelofibrosis: 2014 update on diagnosis, riskstratification, and management. Am J Hematol 2014; 89: 916e25.
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50 Lefevre G, Copin M-C, Staumont-Salle D, et al. The lymphoid variant of hypereosinophilic syndrome. Medicine 2014; 93: 255e66.
41 Wang SA, Hasserjian RP, Fox PS, et al. Atypical chronic myeloid leukemia is clinically distinct from unclassifiable myelodysplastic/myeloproliferative neoplasms. Blood 2014; 123: 2645e51. 42 Meggendorfer M, Haferlach T, Alpermann T, et al. Specific molecular mutation patterns delineate chronic neutrophilic leukemia, atypical chronic myeloid leukemia, and chronic myelomonocytic leukemia. Haematologica 2014; 99: e244e6. 43 Elliott MA, Tefferi A. Chronic neutrophilic leukemia 2014: update on diagnosis, molecular genetics, and management. Am J Hematol 2014; 89: 652e8. 44 Castelli R, Cugnoa M, Gianelli U, et al. Neutrophilic progression in a case of polycytemia vera mimicking chronic neutrophilic leukemia: clinical and molecular characterization. Pathol Res Prac 2015; 211: 341e3. 45 Kohmura K, Miyakawa Y, Kameyama K, et al. Granulocyte colony stimulating factor-producing multiple myeloma associated with neutrophilia. Leuk Lymphoma 2004; 45: 1475e9. 46 Blombery P, Kothari J, Yong K, et al. Plasma cell neoplasm associated chronic neutrophilic leukemia with membrane proximal and truncating CSF3R mutations. Leuk Lymphoma 2014; 55: 1661e2. 47 Erber W, Reilly JT. Chronic neutrophilic leukemia with plasma cell dyscrasia: friends or relatives? Leuk Lymph 2014; 55: 240e2. 48 Tefferi A, Thiele J, Vannucchi AM, et al. An overview on CALR and CSF3R mutations and a proposal for revision of WHO diagnostic criteria for myeloproliferative neoplasms. Leukemia 2014; 28: 1407e13. 49 Gotlib J. World Health Organization-defined eosinophilic disorders: 2014 update on diagnosis, risk stratification, and management. Am J Hematol 2014; 89: 325e37.
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Practice points C
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The classification of BCR-ABL 1 negative myeloproliferative neoplasms must take into account clinical, haematological, morphological and genetic features Myeloproliferative neoplasms exhibit a spectrum of evolution from a prodromal or initial phase, through a stable phase, to a terminal phase, which results in bone marrow failure, myelodysplastic changes and blast crisis Prodromal stages may not present with the classic clinicopathological criteria for diagnosis and follow-up may be required before reaching a definitive diagnosis Follow-up biopsies are important to monitor progression and features of regression, stability, progression and the effects of therapy must be noted Quantitation of fibrosis is very important and it must be graded accurately throughout the course of the disease according to accepted definitions The last decade has seen tremendous advances in molecular biology, diagnosis and treatment of BCR-ABL1-neg MPN, including JAK2V617F and JAK2 exon 12, MPL, CALR (exon 9 deletions and insertions), CSF3R and SETBP1 mutations These advances have led to re-examination of diagnostic criteria and new therapeutic options
Ó 2015 Elsevier Ltd. All rights reserved.
An update on BCR-ABL1negative myeloproliferative neoplasms
PDGFRA, PDGFRB or FGFR1, will not be considered here as the diagnostic process is different. Myeloproliferative neoplasms are clonal haemopoietic stem cell disorders, characterized by proliferation of one or more of the myeloid cell lineages and lack of significant dysplasia. Classification continues to be influenced by recent discoveries of genetic abnormalities involved in the pathogenesis of BCR-ABL1negative MPNs, correlated with specific histological features and clinical findings.
Jon D van der Walt
Abstract BCR-ABL 1 negative myeloproliferative neoplasms (MPN) are clonal haemopoietic stem cell disorders, characterized by proliferation of one or more of the myeloid cell lineages. Classification is based on the genetic abnormalities involved in the pathogenesis and on correlation of specific histological features with clinical findings. The diagnostic approach must take into account clinical, haematological, morphological and genetic features. A clinico-pathologically assigned diagnosis therefore forms the basis of the current consensus based classification. Myeloproliferative neoplasms may have an insidious onset and variable progression, comprising the full spectrum of evolution from a prodromal or initial phase, through a stable phase, to a terminal phase. The latter results in bone marrow failure, myelodysplastic changes and blast crisis. Prodromal stages pose a number of diagnostic problems because they may not present with the classic clinico-pathological criteria for diagnosis. Follow-up biopsies are important to monitor progression and features of regression, stability, progression and the effects of therapy must be noted. In the MPN group particularly, quantitation of fibrosis is very important and it must be graded accurately throughout the course of the disease. The effect of therapies must be taken into account in initial and follow-up biopsies. The last decade has seen tremendous advances in molecular biology, diagnosis and treatment of BCR-ABL1-neg MPN. These advances have led to re-examination of diagnostic criteria and revision of the 2008 WHO classification is required urgently.
Approach to diagnosis The diagnostic approach must take into account clinical, haematological, morphological and genetic features (Box 1). A clinico-pathologically assigned diagnosis forms the basis of the current consensus classification (Box 2). This classification is not without controversy and alternative views have been expressed.2 Reproducibility of the histopathology of certain aspects of the classification also remains controversial. Myeloproliferative neoplasms may have an insidious onset and variable progression, comprising the full spectrum of evolution from a prodromal or initial phase, through a stable phase, to a terminal phase. The latter results in bone marrow failure, myelodysplastic changes and blast crisis (Table 1 ). Prodromal stages pose a number of diagnostic problems because they may not present with the classic clinicopathological criteria for diagnosis.3,4 They may be recognisable by bone marrow examinations but may also result in an “unclassifiable” diagnosis. Rebiopsy may eventually clarify the situation but these cases may initially have to be placed in the MPN, U or MDS/MPN, U category awaiting follow-up investigations and eventual reclassification. Likewise, the onset of ‘acceleration’ and acute transformation is sometimes difficult to define and there is a lack of consensus on diagnostic criteria. The WHO criteria of 20% blasts in blast crisis and 10e19% blasts in accelerated phase may be difficult to apply in practice in cases of ‘dry tap’ marrow aspiration and discordant bone marrow and peripheral blood findings. It is in any case arbitrary. A blast crisis may be the result of clonal evolution that is obvious from an increasingly complex karyotype. During the phase of progression, transition to an MDS/MPN phenotype preceding transformation is often parallelled by development of a complex karyotype typical for secondary AML.5 It should also be noted that extramedullary blast proliferation indicates blast phase and that the marrow findings may be discordant. Follow-up biopsies are important to monitor progression and features of regression, stability, progression and the effects of therapy must be noted (Table 2 ). In the MPN group particularly, quantitation of fibrosis is very important and it must be accurately graded (Box 3). The effects of therapies such as hydroxycarbamide must be taken into account. The differential diagnosis in MPN includes other members of this category, the related refractory anaemia with ring sideroblasts with thrombocytosis (RARS-T)6 and also entities such as MPN/MDS and MDS with isolated del(5q), which may present with thrombocytosis.7 A variety of reactive disorders must be actively excluded before a diagnosis of MPN or MDS/MPN can be established. Of particular interest are the hereditary
Keywords chronic eosinophilic leukaemia; chronic neutrophilic leukaemia; essential thrombocythaemia; myeloproliferative neoplasm; polycythaemia vera; primary myelofibrosis
Introduction In the fourth edition of the WHO classification,1 the term ‘chronic myeloproliferative disease’ was replaced by ‘myeloproliferative neoplasm’ (MPN). Inclusion of systemic mastocytosis (SM) has attracted controversy and, together with the myeloid and lymphoid neoplasms with eosinophilia and abnormalities of
Abbreviations: MPN, myeloproliferative neoplasm; MPN, U, myeloproliferative neoplasm, unclassifiable; MDS, myelodysplastic syndromes; MPN/MDS, myeloproliferative neoplasm/myelodysplastic syndrome; PV, polycythaemia vera; PMF, primary myelofibrosis; ET, essential thrombocythaemia; CML, chronic myelogenous leukaemia; CNL, chronic neutrophilic leukaemia; CEL, chronic eosinophilic leukaemia; AML, acute myeloid leukaemia; CMML, chronic myelomonocytic leukaemia; SM, systemic mastocytosis. Jon D van der Walt MB BCH FRCPATH, Consultant Histopathologist Department of Histopathology, Guy’s and St Thomas’ Hospitals, London, UK. Conflicts of interest: none declared.
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myeloproliferative disorders,8 hereditary thombocytosis9 and congenital erythrocytosis.10,11 Knowledge of the family history and appreciation of variant morphology are crucial for the diagnosis. With the decline in biopsy rates for PV in the post JAK2V617F era, a higher proportion of cases of congenital or acquired erythrocytosis are likely to encountered. Reactive conditions such as infection with HIV12 may cause thrombocytosis. The use of thrombopoietin receptor agonists in thrombocytopenic states13 may mimic ET or PV, with increased reticulin and MPN-like morphology.
Guidelines for using the revised WHO classification of myeloid neoplasms26 Specimen requirements C PB and BM specimens collected prior to any definitive therapy. C PB and cellular BM aspirate smears and/or touch preparations stained with Wright-Giemsa or similar stain. C BM biopsy specimen, at least 1.5 cm in length and at right angles to the cortical bone, is recommended for all cases if feasible. C BM specimens for complete cytogenetic analysis and, when indicated, for flow cytometry, with an additional specimen cryopreserved for molecular genetic studies. The latter studies should be performed based on initial karyotypic, clinical, morphologic, and immunophenotypic findings.
Advances in the molecular pathology of MPN Acquired somatic mutations of JAK2V617F and JAK2 exon 12 have been shown to play a crucial role in the pathogenesis of many cases of BCR-ABL1-negative MPN and these discoveries opened a new era in the understanding of the biology and therapy of MPN.14,15 JAK2V617F mutation is detected in about 95% of patients with PV as a secondary genetic event that is preceded by an as yet undefined molecular abnormality. As a consequence of this mutation, transformation and proliferation of haematopoietic progenitor cells are promoted by downstream signal transduction pathways. In ET and PMF, this mutation is found in only about 50% of patients and in JAK2V617F negative PV, an activating JAK2 exon 12 mutation is detectable.16 In a small number of patients with ET and PMF, an activating mutation of MPLW515L/K is present.17 It must be noted that JAK 2V617F is not specific for MPN and its absence does not exclude the diagnosis. In 2013, two groups independently reported novel calreticulin gene (CALR) mutations (exon 9 deletions and insertions) in JAK2 or MPL unmutated PMF and ET.18,19 In one of the studies, among 1107 patient samples with MPN, CALR mutations were not seen in PV but were seen in ET (25%) and PMF (35%).19 CALR mutations were mutually exclusive of JAK2 or MPL mutations. When JAK2/MPL-unmutated cases were analysed, CALR mutational were found to be 67% in ET and 88% in PMF. CALR mutations were not detected in AML, MDS, CMML or CML. Three patients (13%) with RARS-T displayed CALR mutations and all three were JAK2/MPL-negative. Subsequent studies of CML cases have shown concurrent CALR and BCR-ABL1; these have been described20,21 as ‘ET evolving to CML’ and ‘A CALR Mutation Preceding BCR-ABL1 in an Atypical Myeloproliferative Neoplasm’. These complex cases require synthesis of haematological, morphological and molecular findings to reach a diagnosis. Other mutations, including TET2, IDH, ASXL1 or DNMT3A, are occasionally seen in PV and ET.22 The presence or absence of the various mutations has been found to have prognostic implications. In a recent study of PMF, CALR mutations were mutually exclusive of JAK2 or MPL mutations and were associated with younger age and higher platelet counts, with patients being less likely to be anaemic, require transfusions or display leucocytosis. CALR mutations had a favourable impact on survival while ‘CALR-ASXL1þ’ and ‘triple-negative’ cases were identified as high-risk molecular signatures in PMF.23 In another study, ET patients with CALR mutation had lower leucocyte counts and ages compared with JAK2-mutated ET patients.24 Thus, evaluation of JAK2, MPL, and CALR mutation status has become mandatory not only for diagnosis but also for prognostication.25
Assessment of blasts C Blast percentage in PB and BM is determined by visual inspection. C Myeloblasts, monoblasts, promonocytes, megakaryoblasts (but not dysplastic megakaryocytes) are counted as blasts when summing blast percentage for diagnosis of AML or blast transformation; count abnormal promyelocytes as “blast equivalents” in APL. C Proerythroblasts are not counted as blasts except in rare instances of “pure” acute erythroleukaemia. C Flow cytometric assessment of CD34þ cells is not recommended as a substitute for visual inspection; not all blasts express CD34, and artifacts introduced by specimen processing may result in erroneous estimates. C If the aspirate is poor and/or marrow fibrosis is present, immunohistochemistry (IHC) on biopsy sections for CD34 may be informative if blasts are CD34þ. Assessment of blast lineage C Multiparameter flow cytometry (at least three colours) is recommended; the panel should be sufficient to determine lineage as well as aberrant antigen profile of a neoplastic population. C Cytochemistry, such as myeloperoxidase or nonspecific esterase, may be helpful, particularly in AML, NOS, but it is not essential in all cases. C IHC on biopsy sections may be helpful; many antibodies are now available for recognition of myeloid and lymphoid antigens. Assessment of genetic features C Complete cytogenetic analysis from BM at initial diagnosis when possible. C Additional studies, such as FISH, RT-PCR, mutational status, should be guided by clinical, laboratory, and morphologic information. C Mutational studies for mutated NPM1, CEBPA, and FLT3 are recommended in all cytogenetically normal AML; mutated JAK2 should be sought in BCR-ABL1enegative MPN, and mutational analysis for MPL, CALR, KIT, NRAS, PTNP11, etc, should be performed as clinically indicated. Correlation/reporting of data C All data should be assimilated into one report that states the WHO diagnosis. Box 1
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WHO 2008 characterisation of BCR-ABL1 negative MPN1
Minor criteria C Leukoerythroblastosis C Increase in serum lactate dehydrogenase level C Anaemia C Palpable splenomegaly
Polycythaemia Vera (PV) Figure 1aed . Diagnosis requires the presence of both major criteria and one minor criterion or the presence of the first major criterion and two minor criteria. Major criteria C Hgb >18.5 g/100 ml in men or 16.5 g/100 ml in women or other evidence of increased red cell volume. (Hgb or Hct >99th percentile of reference range for age/sex/altitude or Hgb >99th percentile of reference range for age/sex/altitude or Hgb >17 g/ 100 ml in men or 15 g/100 ml in women if associated with a documented and sustained increase of at least 2 g/100 ml from an individual’s baseline value that is not attributed to correction of iron deficiency anaemia, or elevated red cell mass (>25% above mean normal predicted value.). C Presence of the JAK 2V617F or other functionally similar mutations, such as the JAK2 exon 12 mutation. Minor criteria C BM showing hypercellularity for age with trilineage growth (panmyelosis) and with prominent erythroid and megakaryocytic proliferation. C Serum Epo level below the reference range for normal. C Endogenous erythroid colony formation in vitro
Chronic neutrophilic leukaemia (CNL) C Peripheral blood leucocytosis 25 109/Litre C Hypercellular bone marrow biopsy C Mature neutrophils C No dysplasia C Hepatosplenomegaly C Clonality established or reactive causes excluded C No BCR-ABL1 fusion gene C No rearrangement of PDGFRA, PDGFRB or FGFR1 C No evidence of PV, PMG or ET C No evidence of MDS or MDS/MPN C Slowly progressive
Chronic eosinophilic leukaemia (CEL) C Eosinophilia (1.5 109/Litre) C No Ph chromosome, no BCR-ABL1 fusion gene C No evidence of other MPN, MDS/MPN, AML C No rearrangement of PDGFRA, PDGFRB or FGFR1 C Clonal cytogenetic or molecular genetic abnormality or blasts >2% in PB or >5% in BM. C Blast count <20% C Immunophenotypically aberrant T-lymphocytosis is excluded
Essential thrombocythaemia (ET) Figure 2a . Diagnosis requires meeting all 4 criteria. C C
C
C
Sustained platelet count 450 109/Litre* Bone marrow biopsy specimen showing proliferation mainly of the megakaryocytic lineage with increased numbers of enlarged, mature megakaryocytes. No significant increase or left-shift of neutrophil granulopoiesis or erythropoiesis. Not meeting WHO criteria for polycythemia vera, primary myelofibrosis, BCR-ABL1epositive CML, or MDS or other myeloid neoplasm. Demonstration of JAK 2V617F or other clonal marker, or in the absence of JAK 2V617F, no evidence of reactive thrombocytosis.
Myeloproliferative neoplasm, unclassifiable (MPN, U) C Definite clinical, laboratory or morphological features of MPN C Does not meet criteria for specific MPN or has overlapping features C No BCR-ABL1 fusion gene C No rearrangement of PDGFRA, PDGFRB or FGFR1 C Not used for inadequately investigated cases C Reactive or post therapeutic changes must be excluded C Prodromal or late phase MPN cases are most common
Primary myelofibrosis (PMF) Figure 2bed. Diagnosis requires meeting all 3 major criteria and 2 minor criteria. Major criteria C Presence of megakaryocyte proliferation and atypia,* usually accompanied by either reticulin or collagen fibrosis, or, in the absence of significant reticulin fibrosis, the megakaryocyte changes must be accompanied by an increased bone marrow cellularity characterized by granulocytic proliferation and often decreased erythropoiesis (ie, prefibrotic cellular-phase disease). C Not meeting WHO criteria for PV, CML, MDS, or other myeloid disorders. C Demonstration of JAK 2V617F or other clonal marker (eg, MPLW515K/ L ), or, in the absence of the above clonal markers, no evidence that bone marrow fibrosis is secondary to infection, autoimmune disorder or other chronic inflammatory condition, hairy cell leukemia or other lymphoid neoplasm, metastatic malignancy, or toxic (chronic) myelopathies.
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Box 2
Prodromal states Controversy has long surrounded the diagnosis of cases of MPN that, at presentation, do not manifest all of the classical diagnostic findings but which may subsequently progress. While the diagnosis of overt PV is usually straightforward, particularly since the establishment of JAK2617VF or other functionally similar mutation such as JAK2 exon 12 mutation as a major diagnostic criterion, prodromal-latent stage or socalled ‘masked’ PV may be difficult to assess. Recent proposals26 have called for reduction in the defining haemoglobin levels in the WHO classification, or the introduction of haematocrit as a diagnostic criterion, as proposed by the British Committee for Standards in Haematology.27 The value of bone marrow biopsy is emphasized in these difficult cases of MPN26
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Staging of myelofibrosis (MF) in MPN33
Diagnosis: The three phases of MPN Evolution
Stability
Progression
Criteria incomplete Diagnostic confusion
Classic morphology
Transitional criteria
Criteria well defined
Morphological changes Cytogenetic changes
Cytogenetics stable Diagnostic problems Reactive vs MPN Variant morphology & cytogenetics MPN, U
MF 1 (Reticulin, focal, patchy, endophytic bone formation absent) C Loose network of reticulin with intersections, especially in peripheral areas, no collagenisation. C Sinusoidal Haematopoiesis is absent.
MF 2 (Reticulin plus collagen, diffuse meshwork, endophytic bone formation may be present) C Diffuse increase in reticulin with extensive intersections and only focal bundles of collagen. Hypercellular bone marrow. C Sinusoidal haematopoiesis may be present.
Accelerated vs acute phase Quantitation of fibrosis
MF3 (Mostly collagen, diffuse meshwork, scarring, dilated sinuses, endophytic bone formation scant to prominent) C Diffuse and dense increased reticulin with extensive intersections with course bundles of collagen and significant osteosclerosis. C Sinusoidal haematopoiesis may be prominent.
ET vs pf-PMF PV vs MPN, U Table 1
and also in the distinction from cases of erythrocytosis, in which the classical morphology of MPN is lacking.10,11 It has been noted that the presence of a CALR mutation excludes a diagnosis of PV.28 Some authors have stressed the importance of so called prefibrotic PMF, which can mimic ET in its presentation. The mutation profile is similar for JAK2, CALR and MPL mutations according to these authors.29 It is emphasized that careful morphologic examination of bone marrow histology is necessary to distinguish the two; megakaryocytes in ET being large and mature-appearing whereas those in prefibrotic PMF display abnormal maturation with hyperchromatic and irregularly folded nuclei.4 The distinction between ET and prefibrotic PMF is claimed to be prognostically relevant.30 However, other authors have disputed the reproducibility of the diagnosis.31,32 Clarification is expected in the revised WHO classification.
Box 3
criteria34 in order to establish the point in the progression of MPN at which myelofibrosis develops. It should be noted that it is essential to document a previous diagnosis of ET or PV before confirming a diagnosis of post-PV or post-ET fibrosis. Distinction from PMF may be impossible if a well-documented preceding diagnosis, preferably with biopsy confirmation, is not available (Box 4). Risk stratification according to mutation status and other factors, and management options, have recently been reviewed.29,35 A recent large study36 has shown that the newly discovered mutations have yet to render superfluous the need for morphologic distinction of ET from PV both diagnostically and prognostically and that determination of JAK2/CALR mutational status alone, without morphologic examination, did not differentiate PV from JAK2 mutant ET, while distinguishing morphologically defined ET into JAK2 and CALR subtypes carried limited prognostic relevance. It has recently been recognized that the development of absolute monocytosis in patients with established PMF is associated with rapid disease progression and these patients have a shorter survival.37
Fibrosis and progression All MPN are prone to develop fibrosis and accurate assessment of grade of reticulin fibrosis33 is fundamental to the assessment of bone marrow histology in these patients, at time of diagnosis and on follow-up biopsies. It is necessary to adhere to strictly defined
Chronic neutrophilic leukaemia
Diagnosis: Assessment of follow-up biopsy samples in MPN and MDS/MPN Regression
Stability
Progression
Therapy
Reticulin reduced
Diagnosis unchanged or clarified Reticulin & cellularity stable
Reticulin increased
Normalisation
Spent phase
Cellularity reduced
Acute phase
Dysplasia Acute phase
Cellularity reduced
The current WHO diagnostic criteria for CNL (Table 1) were confounded by the absence of a clonal marker and defining bone marrow morphology. A relatively high leucocyte count threshold was required and reactive granulocytosis, plasma cell neoplasmassociated neutrophilia, CML, aCML and CMML thus had to be excluded. Recently, recurrent somatic mutations in the proximal domain of the gene for colony-stimulating factor 3 receptor (CSF3R) were reported in patients with CNL.38,39 CSF3R was mutated in 100%, SETBP1 in 33%38 and CALR in 12.5% of WHOdefined cases of CNL.40 The original publications38 suggested that somatic CSF3RT618I mutations were present in CNL and aCML. A subsequent
Table 2
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Figure 1 a) PV showing classical panmyelosis and polymorphic megakaryocytes. b) PV with JAK2 exon 12 mutation showing lack of megakaryocyte clustering. c). PV showing progression to grade 2 myelofibrosis. d). PV showing progression to acute myeloid leukaemia.
Figure 2 a). Classical ET showing large mature megakaryocytes with ‘staghorn nuclei’. b). Cellular phase of PMF showing hyperchromatic, abnormal folded nuclei. c). Grade 3 osteomyelofibrosis showing typical megakaryocyte morphology. d). Post hydroxycarbamide changes. A morphological diagnosis of MPN type in not possible.
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manifestations mimicking CNL.45 However, cases of proven CNL with concurrent plasma cell neoplasia have also been described.46 The mechanism by which CNL occurs in association with a plasma cell neoplasm remains unknown and a coincidence is possible.47 Cases of patients with CNL receiving therapy with the JAK1/2 inhibitor ruxolitinib and showing a clinical response have been reported, opening the door to a new therapeutic approach.38 Progression in CNL is not as well defined as in the more common MPN categories but therapy-resistant progressive neutrophilia, thrombocytopenia, splenomegaly, clonal evolution and myeloid blast crisis are recognized.43 Revision of the current WHO classification is expected to include the molecular criterion of mutated CSF3R in the diagnostic criteria for CNL.43,45 A recent proposal principally includes peripheral blood leucocytosis 13 109/litre, >80% neutrophils or bands and the presence of CSF3RT618I or other membrane proximal CSF3R mutations and lack of dysgranulopoiesis. There must be no monocytosis, molecular evidence of BCR-ABL-1, PDGFRA, PDGFRB, or FGRF1 rearrangement.48
Recommended criteria for post-polycythemia vera and post-essential thrombocythemia myelofibrosis34 Criteria for post-polycythemia vera myelofibrosis Required criteria: C
C
Documentation of a previous diagnosis of polycythemia vera as defined by the WHO criteria Bone marrow fibrosis grade 2e3 (on 0e3 scale) or grade 3e4 (on 0e4 scale)
Additional criteria (two are required): C
C C
C
Anaemia or sustained loss of requirement for phlebotomy in the absence of cytoreductive therapy A leucoerythroblastic peripheral blood picture Increasing splenomegaly defined as either an increase in palpable splenomegaly of 5 cm (distance of the tip of the spleen from the left costal margin) or the appearance of a newly palpable splenomegaly Development of 1 of three constitutional symptoms: >10% weight loss in 6 months, night sweats, unexplained fever (>37.5 C)
Chronic eosinophilic leukaemia In contrast to CNL, CEL still remains a diagnosis of exclusion. Secondary eosinophilia has numerous causes and infections, allergic reactions, autoimmune diseases, lymphoma and rare congenital conditions have to be investigated by specialists in different disciplines.49 Once reactive causes are excluded, morphological examination of the bone marrow will be required to consider eosinophilia associated with other WHO entities such as SM, CML, MDS, AML, CMML etc49 and the lymphoid variant of hypereosinophilic syndrome.50 This is defined by the presence of a Th2 T-cell subset overproducing interleukin-5. Many different abnormal T-cell immunophenotypes have been described, the most frequent of which are: CD3 CD4þ, CD3þ CD4þ CD7, and CD3 þ CD4 CD8 TCRabþ. Clonal rearrangement of TCR genes is frequently observed in these abnormal T-cells.50 Investigations should be directed to exclude Philadelphia chromosome or BCR-ABL1 fusion, t(5;12)(q31-q35;p13) or other rearrangement of PDGFRB, FIP1L1-PDGFRA fusion or other rearrangement of PDGFRA and no rearrangement of FGFR1, inv(16)(p13q22) or t(16;16)(p13;q22), or other features diagnostic of AML must be present. Clonal cytogenetics or other abnormal cytogenetics must be established in addition to these exclusions.50
Criteria for post-essential thrombocythemia myelofibrosis Required criteria: C
C
Documentation of a previous diagnosis of essential thrombocythemia as defined by the WHO criteria (see Table 2) Bone marrow fibrosis grade 2e3 (on 0e3 scale) or grade 3e4 (on 0e4 scale)
Additional criteria (two are required): C
C C
C C
Anaemia and a 2 g/dL decrease from baseline haemoglobin level A leucoerythroblastic peripheral blood picture Increasing splenomegaly defined as either an increase in palpable splenomegaly of 5 cm (distance of the tip of the spleen from the left costal margin) or the appearance of a newly palpable splenomegaly Increased lactate dehydrogenase Development of of three constitutional symptoms: >10% weight loss in 6 months, night sweats, unexplained fever (>37.537.5 C)
Box 4
Conclusion publication denied the association with aCML41 but other authors have found it in small number (3%) of patients with aCML.42 The JAK2V617F mutation has also been reported in CNL but its significance is unclear.43 Neutrophilic progression in a case of PV mimicking CNL has been described, emphasizing the need for careful work-up in complex cases.44 CSF3R mutations have also been reported in hereditary chronic neutrophilia.43 There is a well established association between neutrophilia and plasma cell neoplasia. Granulocyte-colony stimulating factor-producing myeloma has been described, with clinical
DIAGNOSTIC HISTOPATHOLOGY 21:5
The last decade has seen tremendous advances in molecular biology, diagnosis and treatment of BCR-ABL1-negative MPN. These advances have led to re-examination of diagnostic criteria and revision of the 2008 WHO classification is required urgently, which is expected in 2016. A REFERENCES 1 Swerdlow SH, Campo E, Harris NL, et al. WHO classification of tumours of haematopoietic and lymphatic tissues. Lyon: IARC, 2008.
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Practice points C
C
C
C
C
C
C
197
The classification of BCR-ABL 1 negative myeloproliferative neoplasms must take into account clinical, haematological, morphological and genetic features Myeloproliferative neoplasms exhibit a spectrum of evolution from a prodromal or initial phase, through a stable phase, to a terminal phase, which results in bone marrow failure, myelodysplastic changes and blast crisis Prodromal stages may not present with the classic clinicopathological criteria for diagnosis and follow-up may be required before reaching a definitive diagnosis Follow-up biopsies are important to monitor progression and features of regression, stability, progression and the effects of therapy must be noted Quantitation of fibrosis is very important and it must be graded accurately throughout the course of the disease according to accepted definitions The last decade has seen tremendous advances in molecular biology, diagnosis and treatment of BCR-ABL1-neg MPN, including JAK2V617F and JAK2 exon 12, MPL, CALR (exon 9 deletions and insertions), CSF3R and SETBP1 mutations These advances have led to re-examination of diagnostic criteria and new therapeutic options
Ó 2015 Elsevier Ltd. All rights reserved.