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WHO Classification of Tumours
Introduction: Myeloid Neoplasms


The WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues (3rd edition) published in 2001 reflected a paradigm shift in the approach to classification of myeloid neoplasms
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Jaffe ES, Harris NL, Stein H, Vardiman JW (Eds.)
World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues.
3rd Edition
IARC Press: Lyon 2001



. For the first time, genetic information was incorporated into diagnostic algorithms provided for the various entities. The publication was prefaced with a comment predicting future revisions necessitated by rapidly emerging genetic information. The current revision is a commentary on the significant new molecular insights that have become available since the publication of the last classification.

The first entity described in this monograph, chronic myelogenous leukaemia (CML) remains the prototype for the identification and classification of myeloid neoplasms. This leukaemia is recognized by its clinical and morphologic features, and its natural progression is characterized by an increase in blasts of myeloid, lymphoid or mixed myeloid/lymphoid ­immunophenotype. It is always associated with the BCR-ABL1 fusion gene that results in the production of an abnormal protein tyrosine kinase (PTK) with enhanced enzymatic activity. This protein is sufficient to cause the leukaemia and also provides a target for protein tyrosine ­kinase inhibitor (PTKI) therapy that has prolonged the lives of thousands of patients with this often fatal illness

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Druker BJ, Guilhot F, O'Brien SG, Gathmann I, Kantarjian H, Gattermann N, Deininger MW, Silver RT, Goldman JM, Stone RM, Cervantes F, Hochhaus A, Powell BL, Gabrilove JL, Rousselot P, Reiffers J, Cornelissen JJ, Hughes T, Agis H, Fischer T, Verhoef G, Shepherd J, Saglio G, Gratwohl A, Nielsen JL, Radich JP, Simonsson B, Taylor K, Baccarani M, So C, Letvak L, Larson RA, (2006)
Five-year follow-up of patients receiving imatinib for chronic myeloid leukemia.
N Engl J Med 355: 2408-17



. This successful integration of clinical, morphologic and genetic information embodies the goal of the WHO classification scheme.

In this revision, a combination of clinical, morphologic, immunophenotypic and ­genetic features is used in an attempt to define disease entities, such as CML, that are biologically homogeneous and clinically relevant —the same approach used in the 3rd edition of the classification. ­Although the previous scheme began to open the door to including genetic abnormalities as criteria to classify myeloid neoplasms, this revision firmly acknowledges that as in CML, recurring genetic abnormalities provide not only objective criteria for recognition of specific entities but also identification of abnormal gene products or pathways that are potential targets for therapy. One example in this revised scheme is the addition of a new subgroup of myeloid neoplasms (Table 1.01) associated with eosinophilia and chromosomal abnormalities that involve the platelet-derived growth factor receptor alpha (PDGFRA) or platelet derived growth factor receptor beta (PDGFRB) genes —a subgroup defined largely by genetic events that lead to constitutive activation of the receptor tyrosine kinase, PDGFR, and that respond to PTKI therapy

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Bain BJ, Fletcher SH (2007)
Chronic eosinophilic leukemias and the myeloproliferative variant of the hypereosinophilic syndrome.
Immunol Allergy Clin North Am 27: 377-88




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Cools J, DeAngelo DJ, Gotlib J, Stover EH, Legare RD, Cortes J, Kutok J, Clark J, Galinsky I, Griffin JD, Cross NC, Tefferi A, Malone J, Alam R, Schrier SL, Schmid J, Rose M, Vandenberghe P, Verhoef G, Boogaerts M, Wlodarska I, Kantarjian H, Marynen P, Coutre SE, Stone R, Gilliland DG (2003)
A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome.
N Engl J Med 348: 1201-14




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Golub TR, Barker GF, Lovett M, Gilliland DG (1994)
Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation.
Cell 77: 307-16



. Similar examples are found throughout the classification in each major subgroup, and include not only neoplasms associated with microscopically recognizable chromosomal abnormalities but also with gene mutations without a cytogenetic correlate as well. On the other hand, the importance of careful clinical, morphological and immunophenotypic characterization of each myeloid neoplasm and correlation with the genetic findings cannot be over-­emphasized. The discovery of activating JAK2 mutations has revolutionized the ­approach to the diagnosis of the myeloproliferative neoplasms (MPN)
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Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S, Vassiliou GS, Bench AJ, Boyd EM, Curtin N, Scott MA, Erber WN, Green AR, (2005)
Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders.
Lancet 365: 1054-61




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James C, Ugo V, Le Couédic JP, Staerk J, Delhommeau F, Lacout C, Garçon L, Raslova H, Berger R, Bennaceur-Griscelli A, Villeval JL, Constantinescu SN, Casadevall N, Vainchenker W (2005)
A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera.
Nature 434: 1144-8




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Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC (2005)
A gain-of-function mutation of JAK2 in myeloproliferative disorders.
N Engl J Med 352: 1779-90




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Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, Wlodarska I, Clark JJ, Moore S, Adelsperger J, Koo S, Lee JC, Gabriel S, Mercher T, D'Andrea A, Fröhling S, Döhner K, Marynen P, Vandenberghe P, Mesa RA, Tefferi A, Griffin JD, Eck MJ, Sellers WR, Meyerson M, Golub TR, Lee SJ, Gilliland DG (2005)
Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis.
Cancer Cell 7: 387-97



. Yet JAK2 mutations are not specific for any single clinical or morphologic MPN phenotype, and are also ­reported in some cases of myelodysplastic syndromes (MDS), myelodysplastic/ myeloproliferative neoplasms (MDS/MPN) and acute myeloid leukaemia (AML). Thus, an integrated, multidisciplinary ­approach is necessary for the classification of myeloid neoplasms.

With so much yet to learn, there may be some "missteps" as traditional approaches to categorization are fused with more ­molecularly-oriented classification schemes. Nevertheless, this revision of the WHO classification is an attempt by the authors, editors and the clinicians who served as members of the Clinical Advisory Committee (CAC) to provide an "evidence-based" classification that can be used in daily practice for therapeutic decisions and yet provide a flexible framework for integration of new data.


> Prerequisites for classification of myeloid neoplasms by WHO criteria

The WHO classification of myeloid neoplasms relies on the morphologic, cytochemical and immunophenotypic features of the neoplastic cells to establish their lineage and degree of maturation and to decide whether cellular proliferation is ­cytologically normal or dysplastic or ­effective or ineffective. The classification is based on criteria applied to initial specimens obtained prior to any definitive therapy, including growth factor therapy, for the myeloid neoplasm. The blast percentage in the peripheral blood, bone marrow and other involved tissues remains of practical importance to categorize myeloid neoplasms and to judge their progression. Cytogenetic and molecular genetic studies are required at the time of diagnosis not only for recognition of specific genetically defined entities, but for establishing a baseline against which future studies can be judged to assess disease progression. Because of the multidisciplinary approach required to diagnose and classify myeloid neoplasms it is recommended that the various diagnostic studies be ­correlated with the clinical findings and ­reported in a single, integrated report. If a definitive classification cannot be reached the report should indicate the reasons why and provide guidelines for additional studies that may clarify the ­diagnosis.

To obtain consistency, the following guidelines are recommended for the evaluation of specimens when a myeloid neoplasm is suspected to be present. It is assumed that this evaluation will be performed with full knowledge of the clinical history and pertinent laboratory data.

Morphology
Peripheral blood: A peripheral blood (PB) smear should be examined and correlated with results of a complete blood count. Freshly made smears should be stained with May-Grünwald-Giemsa or Wright-Giemsa and examined for white blood cell (WBC), red blood cell (RBC) and platelet abnormalities. It is important to ascertain that the smears are well-stained. Evaluation of neutrophil granularity is important when a myeloid disorder is suspected; designation of neutrophils as abnormal based on hypogranular cytoplasm alone should not be considered unless the stain is well-controlled. Manual 200-cell leukocyte differentials of PB smears are recommended in patients with a myeloid neoplasm when the WBC count permits.

Bone marrow aspirate: Bone marrow (BM) aspirate smears should also be stained with May-Grünwald-Giemsa or Wright-Giemsa for optimal visualization of cytoplasmic granules and nuclear chromatin. Because the WHO Classification relies on percentages of blasts and other specific cells to categorize some entities, it is recommended that 500 nucleated BM cells be counted on cellular aspirate smears in an area as close to the particle and as undiluted with blood as possible. Counting from multiple smears may reduce sampling error due to irregular distribution of cells. The cells to be counted include blasts and promonocytes (see definition below), promyelocytes, myelocytes, meta­myelocytes, band neutrophils, segmented neutrophils, eosinophils, basophils, mono­cytes, lymphocytes, plasma cells, erythroid precursors and mast cells. Megakaryo­cytes, including dysplastic forms, are not included. If a concomitant non-myeloid neoplasm is present, such as plasma cell myeloma, it is reasonable to exclude those neoplastic cells from the count used to evaluate the myeloid neoplasm. If an aspirate cannot be obtained due to ­fibrosis or cellular packing, touch preparations of the biopsy may yield valuable cytologic information, but differential counts from touch preparations may not be representative. The differential counts obtained from marrow aspirates should be compared to an estimate of the proportions of cells observed in available biopsy sections.

Bone marrow trephine biopsy: The contribution of adequate BM biopsy sections in the diagnosis of myeloid neoplasms cannot be overstated. The trephine biopsy provides information regarding overall cellularity and the topography, proportion and maturation of haematopoietic cells, and allows evaluation of BM stroma. The biopsy also provides material for immuno­histochemical studies that may have ­diagnostic and prognostic importance. A biopsy is essential whenever there is myelofibrosis, and the classification of some entities, particularly MPN, relies heavily on trephine sections. The specimen must be adequate, taken at right angle from the cortical bone and at least 1.5 cm in length to enable the evaluation of at least 10 partially preserved inter-trabecular areas. It should be well-fixed, thinly sectioned at 3-4 micra, and stained with haematoxylin and eosin and/or a stain such as Giemsa that allows for detailed morphologic evaluation. A silver impregnation method for reticulin fibres is recommended and ­marrow fibrosis graded according to the ­European consensus scoring system

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Thiele J, Kvasnicka HM, Facchetti F, Franco V, van der Walt J, Orazi A (2005)
European consensus on grading bone marrow fibrosis and assessment of cellularity.
Haematologica 90: 1128-32



. A periodic acid-Schiff (PAS) stain may aid in detection of megakaryocytes. Immunohistochemical (IHC) study of the biopsy is often indispensable in the evaluation of myeloid neoplasms and is discussed below.
Fig 1.01 Fig 1.02

Blasts: The percentage of myeloid blasts is important for diagnosis and classification of myeloid neoplasms. In the PB the blast percentage should be derived from a 200-cell leukocyte differential and in the BM from a 500-cell count of cellular BM aspirate smears as described above. The blast percentage derived from the BM ­aspirate should correlate with an estimate of the blast percentage in the trephine biopsy, although large focal clusters or sheets of blasts in the biopsy should be regarded as possible disease progression. Immunohistochemical staining of the BM biopsy for CD34+ blasts often aids in the correlation of aspirate and trephine biopsy findings, although in some myeloid neoplasms the blasts do not express CD34. Flow cytometry determination of blast ­percentage should not be used as a substitute for visual inspection. The specimen for flow cytometry is often haemodilute, and may be affected by a number of pre-analytic variables, and as noted for the biopsy, not all blasts express CD34.

Myeloblasts, monoblasts and megakaryo­blasts are included in the blast count.

Myeloblasts vary from slightly larger than mature lymphocytes to the size of monocytes or larger, with moderate to abundant dark blue to blue-grey cytoplasm. The nuclei are round to oval with finely granular chromatin and usually several nucleoli, but in some nuclear irregularities may be prominent. The cytoplasm may contain a few azurophil granules (Fig 1.03 A B).

Monoblasts are large cells with abundant cytoplasm that can be light grey to deeply blue and may show pseudopod formation (Fig 1.04 top). Their nuclei are usually round with delicate, lacy chromatin and one or more large prominent nucleoli. They are usually strongly positive for non-specific esterase (NSE) but have no or only weak myeloperoxidase (MPO) activity. Promonocytes are considered as "mono­blast equivalents" when the requisite percentage of blasts is tallied for the diagnosis of acute monoblastic, acute monocytic, and acute myelomonocytic leukaemia . Promonocytes have a delicately convoluted, folded or grooved ­nucleus with finely dispersed chromatin, a small, indistinct or absent nucleolus, and finely granulated cytoplasm (Fig 1.04 middle). Most promonocytes express NSE and are likely to have MPO activity. The distinction between monoblasts and promonocytes is often difficult, but ­because the two cell types are summated as monoblasts in making the diagnosis of AML, the distinction between a monoblast and promonocyte is not always critical. On the other hand, distinguishing pro­monocytes from more mature but ­ab­normal leukaemic monocytes can also be difficult, but is critical, because the designation of a case as acute monocytic or acute myelomonocytic leukaemia versus chronic myelomonocytic leukaemia often hinges on this distinction. Abnormal monocytes have more clumped chromatin than a promonocyte, variably indented, folded nuclei and grey cytoplasm with more abundant lilac-colored granules. Nucleoli are usually absent or indistinct (Fig 1.04 lower). Abnormal monocytes are not considered as monoblast equivalents.

Megakaryoblasts are usually of medium to large size with a round, indented or ­irregular nucleus with finely reticular ­chromatin and one to three nucleoli. The cytoplasm is basophilic, usually agranular, and may show cytoplasmic blebs (see acute megakaryoblastic leukaemia ). Small dysplastic megakaryocytes and micromegakaryocytes are not blasts. In acute promyelocytic leukaemia the blast equivalent is the abnormal promyelocyte. Erythroid precursors (proerythroblasts) are not included in the blast count except in the rare instance of "pure"acute erythroid leukaemia in which case they are considered as blast equivalents.

Cytochemistry and other special stains: Cytochemical studies are used to determine the lineage of blasts, although in some laboratories they have been supplanted by immunologic studies using flow cytometry and/or immunohistochemistry. They are usually performed on PB and BM aspirate smears but some can be performed on sections of trephine biopsies or other tissues. Detection of MPO ­indicates myeloid differentiation but its absence does not exclude a myeloid lineage because early myeloblasts as well as monoblasts may lack MPO. The MPO activity in myeloblasts is usually granular and often concentrated in the Golgi region whereas monoblasts, although usually negative, may show fine, scattered MPO+ granules, a pattern that becomes more pronounced in promonocytes. Erythroid blasts, megakaryoblasts and lymphoblasts are MPO negative. Sudan Black B (SBB) staining parallels MPO but is less specific. Occasional cases of lymphoblastic leukaemia exhibit SBB positivity, in which case light grey granules are seen rather than the deeply black granules that characterize myeloblasts. The non-specific esterases, α naphthyl butyrate (ANB) and α naphthyl acetate (ANA), show diffuse cytoplasmic activity in monoblasts and monocytes. Lymphoblasts may have focal punctate activity with NSE but neutrophils are usually negative. Megakaryoblasts and erythroid blasts may have some multifocal, punctate ANA positivity, but it is partially resistant to natrium fluoride (NaF) inhibition whereas monocyte NSE is totally inhibited by NaF. The combination of NSE and the specific esterase, naphthol-ASD-chloroacetate esterase (CAE), which stains primarily cells of the neutrophil lineage and mast cells, permits identification of monocytes and immature and mature neutrophils simultaneously. Some cells, particularly in myelomonocytic leukaemias, may exhibit NSE and CAE ­simultaneously. While normal eosinophils lack CAE, it may be expressed by neoplastic eosinophils. CAE can be performed on tissue sections as well as PB or marrow aspirate smears. In acute erythroid leukaemia, a PAS stain may be helpful in that the cytoplasm of the leukaemic proerythroblasts may show large globules of PAS positivity. Well-­controlled iron stains should always be performed on the BM aspirate to detect iron stores, normal sideroblasts and ring sideroblasts, the latter of which are defined as erythroid precursors with 5 or more granules of iron encircling one-third or more of the ­nucleus.

Immunophenotype
Immunophenotypic analysis using either multiparameter flow cytometry or IHC is an essential tool in the characterization of myeloid neoplasms. Differentiation antigens that appear at various stages of haemato­poietic development and in corresponding myeloid neoplasms are illustrated in Fig. 1.05, and a thorough description of lineage assignment criteria is provided in the chapters on mixed phenotype acute leukaemia. The techniques employed and the antigens analyzed may vary according to the myeloid neoplasm suspected and the information required to best characterize it as well as by the ­tissue available. Although often important in the diagnosis of any haematological neoplasm, immunophenotyping in myeloid neoplasms is most commonly required in AML and in determining the phenotype of blasts at the time of transformation of MDS, MDS/MPN and MPN.

Multiparameter flow cytometry is the ­preferred method of immunophenotypic analysis in AML due to the ability to analyze high numbers of cells in a relatively short period of time with simultaneous recording of information about several antigens for each individual cell. Usually, rather extensive panels of monoclonal antibodies directed against leukocyte differentiation antigens are applied because the utility of individual markers in identifying commitment of leukaemic cells into the different haematopoietic lineages is limited. Evaluation of expression patterns of several antigens, both membrane and cytoplasmic, is necessary for lineage ­assignment, to detect mixed phenotype acute leukaemia, and to detect aberrant phenotypes allowing for follow-up of ­minimal residual disease.

Immunophenotypic analysis has a central role in distinguishing between minimally differentiated acute myeloid leukaemia and acute lymphoblastic leukaemia, and in CML, between myeloid blast phase and lymphoid blast phase. Among AML with recurrent genetic abnormalities, several have characteristic phenotypes. These patterns, described in the respective ­sections, can help to plan molecular cyto­genetic [fluorescence in situ hybridization (FISH)] and molecular investigations in ­individual patients. Immunophenotypic features of the other AML categories are extremely heterogeneous, probably due to high genetic diversity. Although it has been suggested that expression of certain antigens, such as CD7, CD9, CD11b, CD14, CD56 and CD34 could be associated with an adverse prognosis in AML, their independent prognostic value is still controversial. Aberrant or unusual immuno­phenotypes have been found in at least 75% of cases of AML. These can be described as cross-lineage antigen expression, maturational asynchronous ­expres­sion of antigens, antigen overexpression, and the reduction or absence of antigen expression. Similar aberrancies have also been reported in MDS as well, and their presence can be used to support the diagnosis in early or morphologically ambiguous cases of MDS.

Immunophenotyping by IHC on BM biopsy sections can be applied if marrow cell suspensions are not available for flow cytometry analysis. Antibodies reactive with paraffin-embedded BM biopsy tissue are available for many lineage-associated markers (e.g. MPO, lysozyme, CD3, PAX5, CD33, etc.). As noted previously, CD34 staining of the biopsy can facilitate the detection of blasts and their distribution, provided the blasts express CD34

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Orazi A, Neiman RS, Cualing H, Heerema NA, John K (1994)
CD34 immunostaining of bone marrow biopsy specimens is a reliable way to classify the phases of chronic myeloid leukemia.
Am J Clin Pathol 101: 426-8



. For cases rich in megaloblastoid erythro­blasts, immunohistology for glycophorin or haemoglobin may be helpful in distinguishing those cells from myeloblasts (e.g. in cases of refractory anemia with excess blasts (RAEB) or acute erythroid leukaemia ), and CD61 or CD42 often aid in the identification of abnormal mega­karyocytes.

Genetic studies
The WHO classification includes a number of entities defined in part by specific genetic abnormalities, including gene rearrangements due to chromosomal translocations and to specific gene mutations, so determination of genetic features of the neoplastic cells must be performed if possible. A complete cytogenetic analysis of BM should be performed at the time of initial evaluation to establish the cytogenetic profile, and at regular intervals thereafter to detect evidence of genetic evolution. Additional diagnostic genetic studies should be guided by the diagnosis suspected on clinical, morphologic and immunophenotypic studies. In some cases, reverse transcriptase-polymerase chain reaction (RT-PCR) and/or FISH may detect gene rearrangements that are present in low frequency and not observed in the initial chromosomal analysis, in cases with variants of typical cytogenetic abnormalities, and in cases in which the abnormality is cryptic, such as the FIP1L1-PDGFRA fusion in myeloid neoplasms associated with eosinophilia. Depending on the abnormality, quantitative PCR performed at the time of diagnosis may also provide a baseline against which the response to therapy can be monitored. A number of gene mutations detected by gene sequencing, allele-­specific PCR and other techniques have emerged as important diagnostic and prognostic markers in all categories of myeloid neoplasms. Mutations of JAK2, MPL, NRAS, NF1, PTPN11, and KIT in MPN and MDS/MPN, and NPM1, CEBPA, FLT3, RUNX1 and KIT, among others, in AML are important for diagnosis and prognosis, and some, particularly JAK2, FLT3, NPM1 and CEBPA figure importantly in this revised classification. Furthermore, the role of gene over- and under-expression as well as loss of heterozygosity and copy number variants detected by array-based approaches are only now being recognized as important abnormalities that may well influence ­diagnostic and prognostic models in the near future

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Mrózek K, Bloomfield CD (2006)
Chromosome aberrations, gene mutations and expression changes, and prognosis in adult acute myeloid leukemia.
Hematology Am Soc Hematol Educ Program : 169-77



. Nevertheless, ­microarray profiling studies, although ­important in the research setting, have not yet been tested in clinical practice.

> Revised WHO classification of myeloid neoplasms

Table 1.01 lists the major subgroups of myeloid neoplasms and their characteristic features at diagnosis. The nomenclature for the myeloproliferative entities has been changed from "chronic myeloproliferative diseases" to "myeloproliferative neoplasms" and the subgroup formerly designated as "myelodysplastic/myeloproliferative ­diseases" has been changed to "myelodysplastic/myeloproliferative neo­plasms" to underscore their neoplastic nature. Besides the addition of the new subgroup, "Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB and FGFR1," new entities have been added and/or diagnostic criteria updated within each subgroup.

Myeloproliferative neoplasms (MPN)
The MPN (Table 1.02) are clonal haemato­poietic stem cell disorders characterized by proliferation of one or more of the myeloid lineages (i.e. granulocytic, erythroid, megakaryocytic and mast cell). They are primarily neoplasms of adults that peak in frequency in the 5th to 7th decade, but some subtypes, particularly CML and essential thrombocythemia (ET), are reported in children as well. The incidence of all subtypes combined is 6-10/100,000 population annually

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Jemal A, Tiwari RC, Murray T, Ghafoor A, Samuels A, Ward E, Feuer EJ, Thun MJ, (2004)
Cancer statistics, 2004.
CA Cancer J Clin 54: 8-29




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Johansson P (2006)
Epidemiology of the myeloproliferative disorders polycythemia vera and essential thrombocythemia.
Semin Thromb Hemost 32: 171-3




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Johansson P, Kutti J, Andréasson B, Safai-Kutti S, Vilén L, Wedel H, Ridell B (2004)
Trends in the incidence of chronic Philadelphia chromosome negative (Ph-) myeloproliferative disorders in the city of Göteborg, Sweden, during 1983-99.
J Intern Med 256: 161-5



.

Initially, MPN is characterized by hypercellularity of the BM with effective haematopoietic maturation and increased numbers of granulocytes, red blood cells and/or platelets in the PB. Splenomegaly and hepatomegaly are common and caused by sequestration of excess blood cells or proliferation of ­abnormal haemato­poietic cells. Despite an insidious onset each MPN has the ­potential to undergo a stepwise progression that terminates in marrow failure due to myelofibrosis, ineffective haemato­poiesis or transformation to an acute blast phase. Evidence of genetic evolution usually heralds disease progression as may increasing organo­megaly, increasing or decreasing blood counts, myelofibrosis and onset of myelo­dysplasia. The finding of 10-19% blasts in the PB or BM generally signifies accelerated disease and 20% or more is ­sufficient for a diagnosis of blast phase.

Rationale for the diagnosis and classification of MPN
In previous classification schemes the ­detection of the Philadelphia chromosome and/or BCR-ABL1 fusion gene was used to confirm the diagnosis of CML whereas the remaining MPN subtypes were diagnosed by their clinical and ­laboratory features with relatively minor contributions to the diagnosis from morphologic findings. A number of criteria were required not only to distinguish ­subtypes of MPN from each other but from reactive granulocytic, erythroid and/ or megakaryocytic hyperplasia.

Revisions in the criteria for classification of MPN in the current scheme have been influenced by two factors —the recent discovery of genetic abnormalities involved in the pathogenesis of BCR-ABL1 negative MPN and the wider appreciation that histologic features (megakaryocytic morphology and topography, marrow stromal changes, identification of specific cell ­lineages involved in the proliferation) correlate with clinical features and can be used as criteria to identify MPN subtypes

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Tefferi A, Thiele J, Orazi A, Kvasnicka HM, Barbui T, Hanson CA, Barosi G, Verstovsek S, Birgegard G, Mesa R, Reilly JT, Gisslinger H, Vannucchi AM, Cervantes F, Finazzi G, Hoffman R, Gilliland DG, Bloomfield CD, Vardiman JW (2007)
Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel.
Blood 110: 1092-7




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Thiele J, Kvasnicka HM, Orazi A (2005)
Bone marrow histopathology in myeloproliferative disorders--current diagnostic approach.
Semin Hematol 42: 184-95




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Thiele J, Kvasnicka HM, Vardiman J (2006)
Bone marrow histopathology in the diagnosis of chronic myeloproliferative disorders: a forgotten pearl.
Best Pract Res Clin Haematol 19: 413-37



.

Most if not all MPN are associated with clonal abnormalities involving genes that encode cytoplasmic or receptor PTKs. The abnormalities described to date ­include translocations or point mutations of genes that result in abnormal, constitutively abnormal PTKs that activate signal transduction pathways leading to the ­abnormal proliferation. In some cases, these genetic abnormalities, such as the BCR-ABL1 fusion gene in CML, are associated with consistent clinical, laboratory and morphologic findings that allow them to be utilized as major criteria for classification, whereas others provide proof that the myeloid proliferation is neoplastic rather than reactive.

Acquired somatic mutations of JAK2, at chromosome 9p24, have been shown to play a pivotal role in the pathogenesis of many cases of BCR-ABL1 negative MPN

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James C, Ugo V, Le Couédic JP, Staerk J, Delhommeau F, Lacout C, Garçon L, Raslova H, Berger R, Bennaceur-Griscelli A, Villeval JL, Constantinescu SN, Casadevall N, Vainchenker W (2005)
A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera.
Nature 434: 1144-8




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Klion AD, Robyn J, Akin C, Noel P, Brown M, Law M, Metcalfe DD, Dunbar C, Nutman TB (2004)
Molecular remission and reversal of myelofibrosis in response to imatinib mesylate treatment in patients with the myeloproliferative variant of hypereosinophilic syndrome.
Blood 103: 473-8




Click to access Pubmed
Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR, Tichelli A, Cazzola M, Skoda RC (2005)
A gain-of-function mutation of JAK2 in myeloproliferative disorders.
N Engl J Med 352: 1779-90




Click to access Pubmed
Levine RL, Pardanani A, Tefferi A, Gilliland DG (2007)
Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders.
Nat Rev Cancer 7: 673-83




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Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, Wlodarska I, Clark JJ, Moore S, Adelsperger J, Koo S, Lee JC, Gabriel S, Mercher T, D'Andrea A, Fröhling S, Döhner K, Marynen P, Vandenberghe P, Mesa RA, Tefferi A, Griffin JD, Eck MJ, Sellers WR, Meyerson M, Golub TR, Lee SJ, Gilliland DG (2005)
Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis.
Cancer Cell 7: 387-97



. The most common mutation, JAK2 V617F, results in a constitutively active cytoplasmic JAK2 that activates signal transducer and activator of transcription (STAT), mitogen activated protein kinase (MAPK) and phosphotidylinositol 3-kinase (PI3K) signaling pathways to promote transformation and proliferation of haematopoietic progenitors (Fig. 1.07). The JAK2 V617F mutation is found in almost all patients with polycythemia vera (PV) and in nearly one-half of those with primary myelofibrosis (PMF) and with essential thrombocythemia (ET). In the few PV patients who lack the JAK2 V617F, an activating JAK2 exon 12 mutation may be found, and in a small proportion of cases of PMF and ET, an activating mutation of MPL W515L or W515K is seen. It is important to note that JAK2 V617F is not specific for any MPN nor does its absence exclude MPN. Furthermore, it has been reported in some cases of MDS/MPN, in rare cases of AML, and in combination with other well-defined genetic abnormalities such as the BCR-ABL1
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Jones AV, Kreil S, Zoi K, Waghorn K, Curtis C, Zhang L, Score J, Seear R, Chase AJ, Grand FH, White H, Zoi C, Loukopoulos D, Terpos E, Vervessou EC, Schultheis B, Emig M, Ernst T, Lengfelder E, Hehlmann R, Hochhaus A, Oscier D, Silver RT, Reiter A, Cross NC (2005)
Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders.
Blood 106: 2162-8



. Thus, diagnostic algorithms for PV, ET and PMF have been altered to take the mutational status of JAK2 into account as well as to outline the additional laboratory and histologic findings required to reach an accurate classification of cases, regardless of whether the mutation is or is not present.

In addition to the changes in the criteria for PV, ET and PMF, information regarding abnormal PTK function due to rearrangements of the PDGFRA, PDGFRB or FGFR1 genes in patients with myeloid neoplasms associated with eosinophilia led to reappraisal and new diagnostic algorithms for those syndromes as well (see below). The appreciation of the role altered PTKs play in the pathogenesis of CML, PV, ET and PMF also argues for the inclusion of similar chronic myeloid proliferations related to PTK abnormalities under the MPN umbrella. Thus, systemic mastocytosis which has many features in common with other MPN entities and is almost always associated with D816V mutation in the KIT gene encoding the receptor PTK, KIT, has been added to this category

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Tefferi A, Pardanani A (2004)
Clinical, genetic, and therapeutic insights into systemic mast cell disease.
Curr Opin Hematol 11: 58-64



. Still, the molecular pathogenesis of nearly half of all cases of ET and PMF, of all cases of chronic neutrophilic leukaemia and a number of myeloid neoplasms associated with eosinophilia remain unknown. For these reliance on clinical, laboratory and morphologic features is essential for ­diagnosis and classification.

Summary of major changes in the classification of MPN
1. The nomenclature, "myeloproliferative disease" has been changed to "myeloproliferative neoplasm"
2. Mastocytosis has been included in the MPN category
3. Some cases previously meeting the ­criteria for chronic eosinophilic leukaemia (CEL) may now be categorized as myeloid or lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1. If none of these rearrangements are detected, and there is no BCR-ABL1 fusion gene, they should be categorized as chronic eosinophilic leukaemia (CEL), not otherwise specified.
4. The diagnostic algorithms for PV, ET and PMF have been substantially changed to include information regarding JAK2 and similar activating mutations as well as pertinent histologic features of the BM biopsy as diagnostic criteria.
5. The threshold of the platelet count for the diagnosis of ET has been lowered to ≥450x109/L.
6. Criteria for CML in accelerated phase have been suggested with the caveat that they have not been fully evaluated in the era of PTKI therapy; studies to determine their relevance are in progress and revisions may be necessary.

Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1
Determining the cause of marked, persistent eosinophilia (≥1.5x109/L) in the blood can be challenging and is sometimes clinically urgent because of the ­potential damage to the heart, lungs, central nervous and other organ systems caused by the eosinophilic infiltration and release of cytokines, enzymes and other proteins. The eosinophils may be derived from the neoplastic clone of a myeloid neoplasm, such as CEL, CML or AML, or they may be reactive due to abnormal ­cytokine release from reactive or neoplastic T-cells. In a number of cases, no underlying cause can be found and the clonality of the eosinophils cannot be proven; these cases are appropriately termed ­"idiopathic hypereosinophilic syndrome."

Rationale for diagnosis and classification of myeloid and lymphoid disorders with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1
Since the last edition of the WHO classification it has been recognized that many cases of eosinophilia, including a substantial number considered as "idiopathic" are clonal myeloid neoplasms caused by abnormalities in genes that encode the alpha or beta chains of the ­receptor PTKs, platelet derived growth factor receptor (PDGFR) or fibroblast growth factor receptor 1 (FGFR1). Rearrangements of PDGFRB at chromosome band 5q33 that lead to constitutive activation of the beta moiety of PDGFR were first recognized in cases variably reported as CEL or chronic myelomonocytic leukaemia (CMML) with eosinophilia

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Bain BJ, Fletcher SH (2007)
Chronic eosinophilic leukemias and the myeloproliferative variant of the hypereosinophilic syndrome.
Immunol Allergy Clin North Am 27: 377-88




Click to access Pubmed
Golub TR, Barker GF, Lovett M, Gilliland DG (1994)
Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation.
Cell 77: 307-16




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Steer EJ, Cross NC (2002)
Myeloproliferative disorders with translocations of chromosome 5q31-35: role of the platelet-derived growth factor receptor Beta.
Acta Haematol 107: 113-22



. More recently the gene that encodes the alpha moiety of the PDGFR, PDGFRA, at chromosome band 4q12, was found to be ­involved in cryptic translocations in CEL and in nearly one-half of cases reported as idiopathic ­hypereosinophilic syndrome
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Cools J, DeAngelo DJ, Gotlib J, Stover EH, Legare RD, Cortes J, Kutok J, Clark J, Galinsky I, Griffin JD, Cross NC, Tefferi A, Malone J, Alam R, Schrier SL, Schmid J, Rose M, Vandenberghe P, Verhoef G, Boogaerts M, Wlodarska I, Kantarjian H, Marynen P, Coutre SE, Stone R, Gilliland DG (2003)
A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome.
N Engl J Med 348: 1201-14



. In ­addition, rearrangements of the FGFR1 tyrosine kinase gene have also been implicated in myeloproliferations with prominent eosinophilia
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Abruzzo LV, Jaffe ES, Cotelingam JD, Whang-Peng J, Del Duca V, Medeiros LJ (1992)
T-cell lymphoblastic lymphoma with eosinophilia associated with subsequent myeloid malignancy.
Am J Surg Pathol 16: 236-45




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Macdonald D, Reiter A, Cross NC (2002)
The 8p11 myeloproliferative syndrome: a distinct clinical entity caused by constitutive activation of FGFR1.
Acta Haematol 107: 101-7



. However, the clinical and morphologic ­presentations associated with FGFR1 ­rearrangement are variable, and include not only presentation as a myeloproliferative neoplasm with eosinophilia, but also as AML and they may even present as, or evolve to, precursor T or B lymphoblastic leukaemia/lymphoma with prominent eo­sino­phils. Cases associated with PDGFRA rearrangements can likewise present as AML or precursor T-cell neoplasms
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Metzgeroth G, Walz C, Score J, Siebert R, Schnittger S, Haferlach C, Popp H, Haferlach T, Erben P, Mix J, Müller MC, Beneke H, Müller L, Del Valle F, Aulitzky WE, Wittkowsky G, Schmitz N, Schulte C, Müller-Hermelink K, Hodges E, Whittaker SJ, Diecker F, Döhner H, Schuld P, Hehlmann R, Hochhaus A, Cross NC, Reiter A (2007)
Recurrent finding of the FIP1L1-PDGFRA fusion gene in eosinophilia-associated acute myeloid leukemia and lymphoblastic T-cell lymphoma.
Leukemia 21: 1183-8



. Although it might seem most efficient to categorize these cases as CEL within MPN, this would ignore cases with PDGFRB abnormalities that present as CMML as well as cases of FGFR1 and PDGFRA rearrangements that may even have a lymphoid component. To accommodate these translocations, a new subgroup defined largely by the ­genetic abnormalities of PDGFRA, PDGFRB or FGFR1 has been added (Table 1.03). ­Detection of one of these ­abnormalities places the case in this category, regardless of the morphologic classification. Cases of myeloid neoplasms with eosinophilia that lack all of these abnormalities and that meet the criteria for chronic eosinophilic leukaemia, NOS in the MPN category should be placed in that group.

Myelodysplastic/myeloproliferative neoplasms (MDS/MPN)
The MDS/MPN (Table 1.04) include clonal myeloid neoplasms that at the time of initial presentation have some clinical, laboratory or morphologic findings that support a diagnosis of MDS, and other findings more consistent with MPN. They are usually characterized by hypercellularity of the BM due to proliferation in one or more of the myeloid lineages. Frequently, the proliferation is effective in some lineages with increased numbers of circulating cells that may be morphologically and/or functionally dysplastic. Simultaneously, one or more of the other lineages may exhibit ineffective proliferation so that cytopenia(s) may be present as well. The blast percentage in the BM and blood is always <20%. Although ­hepatosplenomegaly is common, the clinical and laboratory findings vary and lie along a continuum between those usually associated with MDS or those usually ­associated with MPN. Patients with a well-defined MPN who develop dysplasia and ineffective haematopoiesis as part of the natural history of their disease or after chemotherapy should not be placed in this category. Rarely, some patients may present in a transformed stage of an MPN entity in which the chronic phase was not recognized, and may have findings that suggest that they belong to the MDS/MPN group. In such cases, if clinical and laboratory studies fail to reveal the nature of the underlying process, the designation of MDS/MPN, unclassifiable may be appropriate. Patients who have the BCR-ABL1 fusion gene or rearrangements of PDGFRA should not be categorized as MDS/MPN, and in contrast to the criteria used in the 3rd edition of the WHO classification, cases of CMML with PDGFRB rearrangements are also excluded.

Rationale for diagnosis and classification of MDS/MPN
This diagnostic category was introduced in the 3rd edition amidst controversy as to whether some entities, particularly CMML, would be better categorized as either MDS or MPN depending on the extent of myeloproliferation as evidenced by the WBC count. Some cases of CMML have low neutrophil counts and only modestly elevated monocyte counts and resemble MDS clinically and morphologically whereas others have markedly elevated WBC counts and organomegaly more in keeping with MPN, yet criteria that clearly distinguish biologically relevant subtypes of CMML remain to be defined.

To date, a few cases of CMML and atypical chronic myeloid leukaemia, BCR-ABL1 negative (aCML) have been reported to demonstrate JAK2 mutations that characterize BCR-ABL1 negative MPN, but the proliferative aspects of most cases of MDS/MPN are related to aberrancies in the RAS/MAPK signaling pathways. In juvenile myelomonocytic leukaemia (JMML) nearly 80% of patients demonstrate ­mutually exclusive mutations of PTNPN11, NRAS or KRAS, or NF1

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Loh ML, Vattikuti S, Schubbert S, Reynolds MG, Carlson E, Lieuw KH, Cheng JW, Lee CM, Stokoe D, Bonifas JM, Curtiss NP, Gotlib J, Meshinchi S, Le Beau MM, Emanuel PD, Shannon KM (2004)
Mutations in PTPN11 implicate the SHP-2 phosphatase in leukemogenesis.
Blood 103: 2325-31




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Stephens K, Weaver M, Leppig KA, Maruyama K, Emanuel PD, Le Beau MM, Shannon KM (2006)
Interstitial uniparental isodisomy at clustered breakpoint intervals is a frequent mechanism of NF1 inactivation in myeloid malignancies.
Blood 108: 1684-9




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Tartaglia M, Niemeyer CM, Fragale A, Song X, Buechner J, Jung A, Hählen K, Hasle H, Licht JD, Gelb BD (2003)
Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia.
Nat Genet 34: 148-50



, all of which encode signaling proteins in RAS dependent pathways, and approximately 30-40% of cases of CMML and aCML exhibit NRAS mutations
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Paquette RL, Landaw EM, Pierre RV, Kahan J, Lübbert M, Lazcano O, Isaac G, McCormick F, Koeffler HP (1993)
N-ras mutations are associated with poor prognosis and increased risk of leukemia in myelodysplastic syndrome.
Blood 82: 590-9




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Vardiman JW (2004)
Myelodysplastic/myeloproliferative diseases.
Cancer Treat Res 121: 13-43




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Willman CL (1998)
Molecular genetic features of myelodysplastic syndromes (MDS)
Leukemia 12 Suppl 1: S2-6



. In view of the lack of any specific genetic abnormality to suggest that these entities should be relocated to another myeloid subgroup, they remain in this "mixed" category which acknowledges the overlap that may occur between MDS and MPN. Cases of CMML with eosino­philia associated with PDGFRB rearrangements are excluded, but rare cases of CMML with eosinophilia that do not exhibit such rearrangements should be classified in this category.

The most controversial issue in the subgroup of MDS/MPN is the provisional entity, refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). The majority (50-60%) of cases of RARS-T studied for JAK2 V617F carry this mutation

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Boissinot M, Garand R, Hamidou M, Hermouet S (2006)
The JAK2-V617F mutation and essential thrombocythemia features in a subset of patients with refractory anemia with ring sideroblasts (RARS).
Blood 108: 1781-2




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Ceesay MM, Lea NC, Ingram W, Westwood NB, Gäken J, Mohamedali A, Cervera J, Germing U, Gattermann N, Giagounidis A, Garcia-Casado Z, Sanz G, Mufti GJ (2006)
The JAK2 V617F mutation is rare in RARS but common in RARS-T.
Leukemia 20: 2060-1




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Gattermann N, Billiet J, Kronenwett R, Zipperer E, Germing U, Nollet F, Criel A, Selleslag D (2007)
High frequency of the JAK2 V617F mutation in patients with thrombocytosis (platelet count>600x109/L) and ringed sideroblasts more than 15% considered as MDS/MPD, unclassifiable.
Blood 109: 1334-5




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Remacha AF, Nomdedéu JF, Puget G, Estivill C, Sarda MP, Canals C, Aventin A (2006)
Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis.
Haematologica 91: 719-20




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Renneville A, Quesnel B, Charpentier A, Terriou L, Crinquette A, Laï JL, Cossement C, Lionne-Huyghe P, Rose C, Bauters F, Preudhomme C (2006)
High occurrence of JAK2 V617 mutation in refractory anemia with ringed sideroblasts associated with marked thrombocytosis.
Leukemia 20: 2067-70




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Schnittger S, Bacher U, Haferlach C, Dengler R, Kröber A, Kern W, Haferlach T (2008)
Detection of an MPLW515 mutation in a case with features of both essential thrombocythemia and refractory anemia with ringed sideroblasts and thrombocytosis.
Leukemia 22: 453-5




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Steensma DP, Caudill JS, Pardanani A, McClure RF, Lasho TL, Tefferi A (2006)
MPL W515 and JAK2 V617 mutation analysis in patients with refractory anemia with ringed sideroblasts and an elevated platelet count.
Haematologica 91: ECR57




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Szpurka H, Tiu R, Murugesan G, Aboudola S, Hsi ED, Theil KS, Sekeres MA, Maciejewski JP (2006)
Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), another myeloproliferative condition characterized by JAK2 V617F mutation.
Blood 108: 2173-81




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Wang SA, Hasserjian RP, Loew JM, Sechman EV, Jones D, Hao S, Liu Q, Zhao W, Mehdi M, Galili N, Woda B, Raza A (2006)
Refractory anemia with ringed sideroblasts associated with marked thrombocytosis harbors JAK2 mutation and shows overlapping myeloproliferative and myelodysplastic features.
Leukemia 20: 1641-4



. This has prompted the notion that RARS-T should be moved to the MPN group of myeloid neoplasms, whereas others have argued that RARS-T is not an entity at all but merely one of the better recognized MPN entities, such as PMF or ET, in which genetic evolution has led to a dysplastic feature, ring sideroblasts
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Schmitt C, Balogh B, Grundt A, Buchholtz C, Leo A, Benner A, Hensel M, Ho AD, Leo E (2006)
The bcl-2/IgH rearrangement in a population of 204 healthy individuals: occurrence, age and gender distribution, breakpoints, and detection method validity.
Leuk Res 30: 745-50




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Szpurka H, Tiu R, Murugesan G, Aboudola S, Hsi ED, Theil KS, Sekeres MA, Maciejewski JP (2006)
Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), another myeloproliferative condition characterized by JAK2 V617F mutation.
Blood 108: 2173-81




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Wang SA, Hasserjian RP, Loew JM, Sechman EV, Jones D, Hao S, Liu Q, Zhao W, Mehdi M, Galili N, Woda B, Raza A (2006)
Refractory anemia with ringed sideroblasts associated with marked thrombocytosis harbors JAK2 mutation and shows overlapping myeloproliferative and myelodysplastic features.
Leukemia 20: 1641-4



. In a few cases reported, however, the cells of patients with RARS-T, when studied by in vitro culture techniques, have growth characteristic more in keeping with MDS than MPN
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Boissinot M, Garand R, Hamidou M, Hermouet S (2006)
The JAK2-V617F mutation and essential thrombocythemia features in a subset of patients with refractory anemia with ring sideroblasts (RARS).
Blood 108: 1781-2




Click to access Pubmed
Remacha AF, Nomdedéu JF, Puget G, Estivill C, Sarda MP, Canals C, Aventin A (2006)
Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis.
Haematologica 91: 719-20



. An additional question is how to clearly distinguish RARS-T from refractory anemia with ring sideroblasts (RARS), in which moderately elevated platelet counts are often reported. This question is more pressing in view of the revised criteria for RARS-T that lowers the platelet threshold from 600x109/L to 450x109/L, in parallel with the revised threshold for ET. It is important to note that the diagnostic criteria for RARS-T include not only the finding of an elevated platelet count in conjunction with anaemia and ring sideroblasts in the BM, but also morphologically abnormal megakaryocytes similar to those of ET or PMF. Only a few patients with RARS and platelet counts in the 450-500x109/L range have been studied for JAK2 mutations and in most with platelet counts in the lower ranges no mutations have been found. Nevertheless, more studies are needed, and we recommend to test for JAK2 mutations in patients who have RARS and platelet counts above the normal range. The sum of current information regarding RARS-T argues for its continued placement in the MDS/MPN category, but in view of the ­debate regarding its precise definition and nature, it is best regarded as a ­"provisional entity" until more data are available.

Lastly, classification of myeloid neoplasms that carry an isolated isochromosome 17q and that have less than 20% blasts in the PB or BM may prove ­difficult. Some authors suggest this cytogenetic defect ­defines a unique disorder characterized by mixed MDS and MPN features associated with prominent pseudo-Pelger-Huët anomaly of the neutrophils, low BM blast count, and a rapidly progressive clinical course. Most cases reported have a prominent monocytic component and meet the criteria for CMML, but in some, the PB monocyte count may not reach the lower threshold for that diagnosis

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Fioretos T, Strömbeck B, Sandberg T, Johansson B, Billström R, Borg A, Nilsson PG, Van Den Berghe H, Hagemeijer A, Mitelman F, Höglund M (1999)
Isochromosome 17q in blast crisis of chronic myeloid leukemia and in other hematologic malignancies is the result of clustered breakpoints in 17p11 and is not associated with coding TP53 mutations.
Blood 94: 225-32




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McClure RF, Dewald GW, Hoyer JD, Hanson CA (1999)
Isolated isochromosome 17q: a distinct type of mixed myeloproliferative disorder/myelodysplastic syndrome with an aggressive clinical course.
Br J Haematol 106: 445-54



. In cases that do not fulfill the criteria for CMML or another well defined myeloid category, designation as MDS/MPN, unclassifiable, with isolated isochromosome 17q abnormality, is most appropriate.

Summary of major changes in MDS/MPN
1. Some cases of CMML with eosinophilia are relocated to the category, "Myeloid neoplasms with PDGFRB rearrangement"
2. The category, "Atypical CML" has been renamed as "Atypical CML, BCR-ABL1 negative" to emphasize this disease is not merely a variant of BCR-ABL1 positive CML.
3. RARS-T remains as a provisional entity, classified as MDS/MPN, unclassifiable, until further data clarifies its appropriate designation. The criteria for its recognition have been modified. The platelet threshold has been lowered to ≥450x109/L, and megakaryocytes with morphology similar to those seen in ET or PMF must be present.

Myelodysplastic syndromes (MDS)
These disorders, usually characterized by the simultaneous proliferation and apoptosis of haematopoietic cells that lead to a normal or hypercellular BM biopsy and PB cytopenia(s), remain among the most challenging of the myeloid neoplasms for proper diagnosis and classification. The general features of MDS, as well as ­specific guidelines for diagnosis and classification are outlined in Overview: Myelodysplastic syndromes/neoplasms

An important addition to the MDS category (Table 1.05) is the provisional entity, refractory cytopenia of childhood (RCC). This category is reserved for children with MDS who have <2% blasts in their PB and <5% in their BM and persistent cytopenia(s) with dysplasia. In contrast to MDS with refractory cytopenias in adults, the majority of cases of RCC have hypocellular BM biopsy specimens, and the distinction from acquired aplastic anaemia and ­inherited BM failure syndromes is often challenging

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Pui CH, Schrappe M, Ribeiro RC, Niemeyer CM (2004)
Childhood and adolescent lymphoid and myeloid leukemia.
Hematology Am Soc Hematol Educ Program : 118-45



.

An issue appropriate to mention at this point is the criteria used for classification of myeloid neoplasms in which 50% or more of the BM cells are erythroid precursors, and for which acute erythroid leukaemia is considered a possible diagnosis. In such cases, if blasts account for fewer than 20% of WBC in the PB and of all nucleated BM cells, and for less than 20% of the non-erythroid cells in the BM (lymphocytes, plasma cells, etc. are also excluded in this latter calculation), the case is considered as MDS. In this scenario, there is lack of consensus among members of the WHO committee as to whether the MDS should then be classified according to the blast percentage of all nucleated BM cells or ­according to the blast percentage of all non-erythroid BM cells, but the majority recommends that the MDS be classified using the blast percentage of all marrow nucleated cells. Many cases of refractory anaemia with ring sideroblasts as well as refractory anaemia have marked erythroid proliferation and using the blast percentage of the non-erythroid cells for classification of such cases might cause these to be placed in an unnecessarily high-risk category. On the other hand, if there is ­severe multilineage dysplasia, very bizarre erythroid morphology, and/or minimal or no maturation to segmented neutrophils, and the blast percentage of total BM cells is not sufficient to place the case into a high-grade MDS category, the case should be flagged for clinical correlation and discussion, with careful follow-up (Table 1.06). More studies are needed however to clarify this controversial issue.

Acute myeloid leukaemia (AML)
AML is a disease resulting from the clonal expansion of myeloid blasts in the PB, BM, or other tissue. It is a heterogeneous disease clinically, morphologically and genetically and may involve only one or all myeloid lineages. Worldwide the incidence is approximately 2.5-3 cases per 100,000 population per year, and is ­reportedly highest in Australia, Western Europe and the United States. The median age at diagnosis is 65 years, with a slight male predominance in most countries. In children less than 15 years of age, AML comprises 15-20% of all cases of acute leukaemia, with a peak incidence in the first 3-4 years of life

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Deschler B, Lübbert M (2006)
Acute myeloid leukemia: epidemiology and etiology.
Cancer 107: 2099-107




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Yamamoto JF, Goodman MT (2008)
Patterns of leukemia incidence in the United States by subtype and demographic characteristics, 1997-2002.
Cancer Causes Control 19: 379-90



.

The requisite blast percentage for a diagnosis of AML is 20% or more myeloblasts and/or monoblasts/promonocytes and/or megakaryoblasts in the PB or BM. The diagnosis of myeloid sarcoma is synonymous with AML regardless of the number of blasts in the PB or BM, unless the ­patient has a prior history of MPN or MDS/MPN, in which case myeloid sarcoma is evidence of acute transformation. The diagnosis of AML can also be made when the blast percentage in the PB and/or BM is less than 20% if there is an associated t(8;21)(q22;q22), inv(16)(p13.1q22), t(16;16)(p13.1;q22) or t(15;17)(q22;q12) chromosomal abnormality, and in some cases of acute erythroid leukaemia when erythroid precursors account for 50% or more of the BM cells and blasts account for 20% or more of the non-erythroid marrow cells.

Although the diagnosis of AML using the above guidelines is operationally useful to indicate an underlying defect in myeloid maturation, the diagnosis does not necessarily translate into a mandate to treat the patient for AML; clinical factors, including the pace of progression of the disease, must always be taken into consideration when deciding therapy.

Rationale for the WHO diagnosis and classification of AML (Table 1.07).
The 3rd edition of the WHO classification ushered in the era of formal incorporation of genetic abnormalities in the diagnostic algorithms for the diagnosis of AML. The abnormalities included were mainly chromosomal translocations involving transcription factors and associated with characteristic clinical, morphologic and immunophenotypic features that formed a "clinico-pathologic-genetic" entity. As knowledge regarding leukaemogenesis has increased, so has the acceptance that the genetic abnormalities leading to leukaemia are not only heterogeneous, but complex, and multiple aberrations often cooperate in a multistep process to initiate the complete leukaemia phenotype. Experimental evidence suggests that in many cases, although rearrangement of genes such as RUNX1, CBFB or RARA that encode transcription factors impair myeloid differentiation, a second genetic abnormality is necessary to promote proliferation or survival of the neoplastic clone (Fig. 1.08)

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Kelly LM, Gilliland DG (2002)
Genetics of myeloid leukemias.
Annu Rev Genomics Hum Genet 3: 179-98



. Often, the additional abnormalities are mutations of genes such as FLT3 or KIT that encode proteins that activate signal transduction pathways to promote proliferation/survival. A similar multistep process is also evident in AML that evolves from MDS or that has myelodysplasia-related features, often characterized by loss of genetic material and haploinsufficiency of genes. Within the last few years, genetic mutations have also been identified in cytogenetically normal AML
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Mrózek K, Marcucci G, Paschka P, Whitman SP, Bloomfield CD (2007)
Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification?
Blood 109: 431-48



. Some of the mutations, such as those of CEBPA and perhaps NPM1 involve transcription factors, whereas others, including those of FLT3 and NRAS/KRAS, affect signal transduction. Not only have these mutations led to an understanding of leukaemogenesis in ­cytogenetically normal AML, but they have proved to be powerful prognostic factors
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Mrózek K, Marcucci G, Paschka P, Whitman SP, Bloomfield CD (2007)
Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification?
Blood 109: 431-48



. In summary, genetic abnormalities in AML elucidate the pathogenesis of the neoplasm, provide the most reliable prognostic information, and will likely lead to development of more successful targeted therapy.

One of the challenges in this revision has been how to incorporate important and/or recently acquired genetic information into a classification scheme of AML and yet adhere to the WHO principle of defining homogeneous, biologically relevant entities based not only on genetic studies or their prognostic value, but also on clinical, morphologic and/or immunophenotypic studies. This was particularly problematic for the most frequent and prognostically important mutations in cytogenetically normal AML, mutated FLT3, NPM1 and CEBPA. They have few or variably consistent morphologic, immunophenotypic and clinical features reported to date, and the mutations are not mutually exclusive.

For the most part, the framework constructed in the 3rd edition proved flexible enough to incorporate the new entities proposed by members of the WHO committee and the CAC (Table 1.07). The entities initially described in the subgroup "AML with recurring genetic abnormalities" remain with only minor modifications (Table 1.07) and three more entities, characterized by chromosomal translocations associated with fairly uniform morphological and clinical features have been added. Cases with mutated NPM1 and CEBPA are added to the same subgroup as "provisional entities" indicating that more study is needed to fully characterize and establish them as unique entities. ­Although mutated FLT3 is not included as a separate entity because it is found to be associated with a number of other entities, its significance should not be underestimated, and it is essential that it be tested for in all cytogenetically normal patients, including those who demonstrate NPM1 and CEBPA mutations.

Modifications have been made in the ­subgroup previously termed "AML with multilineage dysplasia." Initially, it was ­envisioned that this group would encompass biologically unique AML characterized by MDS-like features, including unfavourable cytogenetics, a higher incidence of overexpression of multidrug ­resistance glycoprotein (ABCB1 or MDR-1) and an unfavourable response to therapy. Dysplasia in ≥50% of cells in two or more haematopoietic lineages was used as a universally-applicable surrogate marker for the myelodysplasia-related features. Although the clinical significance of this group has been verified in some studies, it has been disputed in others in which multivariate analysis showed that multilineage dysplasia had no independent significance in predicting clinical outcome when cytogenetic findings were incorporated in the analysis

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Arber DA, Stein AS, Carter NH, Ikle D, Forman SJ, Slovak ML (2003)
Prognostic impact of acute myeloid leukemia classification. Importance of detection of recurring cytogenetic abnormalities and multilineage dysplasia on survival.
Am J Clin Pathol 119: 672-80




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Haferlach T, Schoch C, Löffler H, Gassmann W, Kern W, Schnittger S, Fonatsch C, Ludwig WD, Wuchter C, Schlegelberger B, Staib P, Reichle A, Kubica U, Eimermacher H, Balleisen L, Grüneisen A, Haase D, Aul C, Karow J, Lengfelder E, Wörmann B, Heinecke A, Sauerland MC, Büchner T, Hiddemann W (2003)
Morphologic dysplasia in de novo acute myeloid leukemia (AML) is related to unfavorable cytogenetics but has no independent prognostic relevance under the conditions of intensive induction therapy: results of a multiparameter analysis from the German AML Cooperative Group studies.
J Clin Oncol 21: 256-65




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Wandt H, Schäkel U, Kroschinsky F, Prange-Krex G, Mohr B, Thiede C, Pascheberg U, Soucek S, Schaich M, Ehninger G (2008)
MLD according to the WHO classification in AML has no correlation with age and no independent prognostic relevance as analyzed in 1766 patients.
Blood 111: 1855-61




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Yanada M, Suzuki M, Kawashima K, Kiyoi H, Kinoshita T, Emi N, Saito H, Naoe T (2005)
Long-term outcomes for unselected patients with acute myeloid leukemia categorized according to the World Health Organization classification: a single-center experience.
Eur J Haematol 74: 418-23



. Accordingly, in this revision, this group has been renamed as "acute myeloid leukaemia with myelodysplasia-related changes ." Patients may be assigned to this category if they evolve from previously documented MDS, have specific myelodysplasia-­related cytogenetic abnormalities, or lastly, if they exhibit morphologic multilineage dysplasia as defined above. Patients in this latter group with a normal karyotype should be evaluated for FLT3, NPM1 and CEBPA mutations. Currently, however, the clinical significance of a mutation of one or more of these genes in the setting of morphologic multilineage dysplasia is not clear. Future studies may well prove that such cases are better classified according to their genetic abnormalities, but until more data are available, we recommend that such cases be classified as AML with myelodysplasia-related changes (multilineage dysplasia) with the mutational status of the gene appended.

Therapy-related myeloid neoplasms (t-AML/ t-MDS and t-AML/t-MDS/MPN) remain in the revised classification as a distinct subgroup. However, most patients who develop therapy-related neoplasms have received therapy with both alkylating agents as well as with topoisomerase II ­inhibitors, so that a division according to the type of therapy is usually not practical and not recommended in this revision. It has been argued that 90% or more of cases with t-AML/t-MDS or t-AML/t-MDS/ MPN have cytogenetic abnormalities ­similar to those seen in AML with recurrent genetic abnormalities or AML with myelodysplasia-related features and could be assigned to those categories. However, except for patients with t-AML who have inv(16)(p13.1q22), t(16;16)(p13.1;q22) or t(15;17)(q22;q12), in most reported series those with therapy-related myeloid neoplasms have a significantly worse clinical outcome than their de novo counterparts with the same genetic abnormalities

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Andersen MK, Larson RA, Mauritzson N, Schnittger S, Jhanwar SC, Pedersen-Bjergaard J (2002)
Balanced chromosome abnormalities inv(16) and t(15;17) in therapy-related myelodysplastic syndromes and acute leukemia: report from an international workshop.
Genes Chromosomes Cancer 33: 395-400




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Bloomfield CD, Archer KJ, Mrózek K, Lillington DM, Kaneko Y, Head DR, Dal Cin P, Raimondi SC (2002)
11q23 balanced chromosome aberrations in treatment-related myelodysplastic syndromes and acute leukemia: report from an international workshop.
Genes Chromosomes Cancer 33: 362-78




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Rowley JD, Olney HJ (2002)
International workshop on the relationship of prior therapy to balanced chromosome aberrations in therapy-related myelodysplastic syndromes and acute leukemia: overview report.
Genes Chromosomes Cancer 33: 331-45




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Slovak ML, Bedell V, Popplewell L, Arber DA, Schoch C, Slater R (2002)
21q22 balanced chromosome aberrations in therapy-related hematopoietic disorders: report from an international workshop.
Genes Chromosomes Cancer 33: 379-94




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Smith SM, Le Beau MM, Huo D, Karrison T, Sobecks RM, Anastasi J, Vardiman JW, Rowley JD, Larson RA (2003)
Clinical-cytogenetic associations in 306 patients with therapy-related myelodysplasia and myeloid leukemia: the University of Chicago series.
Blood 102: 43-52



, suggesting some biological differences between the two groups. Furthermore, the study of therapy-related neoplasms may provide valuable insight into the pathogenesis of de novo disease by providing clues as to why a few patients develop leukaemia whereas most patients treated with the same therapy do not. Therefore, patients with therapy-related neoplasms should be designated as such, but the specific cytogenetic abnormality should also be listed, for example, "therapy-­related AML with t(9;11)(p22;q23)".

Acute myeloid leukaemia, NOS Acute myeloid leukaemia, not otherwise specified, encompasses those cases that do not fulfil the specific criteria of any of the other entities. This group accounts for only 25-30% of all AML that are not assigned to one of the more specific categories. As more genetic subgroups are identified, the number of patients that fall into the AML, NOS categories will continue to diminish. Of note is that information used to characterize the subgroups within this category, such as epidemiologic or clinical outcome, is often based on older studies that included patients now assigned to different diagnostic categories, and may not be reliable. Although the proposal to collapse this category into fewer subgroups has been made, the notion that some of these may yet be found to be associated with specific genetic or biologic abnormalities ­argued to maintain this category.

Myeloid sarcoma , an extramedullary ­tumour mass consisting of myeloid blasts, is included in the classification as a distinct pathologic entity. However, when myeloid sarcoma occurs de novo, the ­diagnosis is equivalent to a diagnosis of AML, and further evaluation, including ­genetic analysis, is necessary to determine the appropriate classification of the leukaemia

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Pileri SA, Ascani S, Cox MC, Campidelli C, Bacci F, Piccioli M, Piccaluga PP, Agostinelli C, Asioli S, Novero D, Bisceglia M, Ponzoni M, Gentile A, Rinaldi P, Franco V, Vincelli D, Pileri A, Gasbarra R, Falini B, Zinzani PL, Baccarani M (2007)
Myeloid sarcoma: clinico-pathologic, phenotypic and cytogenetic analysis of 92 adult patients.
Leukemia 21: 340-50



. When the PB and BM is concurrently involved by AML, these tissues may be used for analysis and further classification. However, when the myeloid sarcoma precedes evidence of PB or BM involvement, the immunophenotype should be ascertained by flow cytometry and/or immunohistochemistry, and the genotype determined by cytogenetic analysis, or in the absence of fresh tissue, by FISH or molecular analysis for recurrent genetic abnormalities. Myeloid sarcoma may also be the initial indication of relapse in a ­patient previously diagnosed with AML, or may indicate disease progression to a blast phase in patients with a prior diagnosis of MDS, MDS/MPN or MPN.

Lastly, the unique features of Down syndrome-related myeloid neoplasms has been recognized in a separate listing that includes transient abnormal myelopoiesis and myeloid leukaemias (MDS/AML) ­associated with Down syndrome.(Table 1.07).

Summary of major changes in AML
1. AML with recurrent genetic abnormalities.
a) AML with t(8;21)(q22;q22), AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22), and APL with t(15;17)(q22;q12) are considered as AML regardless of blast count; for all others, blasts ≥20% of PB or of all nucleated BM cells are required.
b) In APL with t(15;17)(q22;q12); PML-RARA, variant RARA translocations with other partner genes are recognized separately; not all have typical APL features and some have ATRA resistance.
c) The former category, AML with 11q23 (MLL) abnormalities has been re-defined as "AML with t(9;11)(p22;q23); MLLT3-MLL". Balanced translocations other than that involving MLLT3 should be specified in the diagnosis. Other abnormalities of MLL, such as partial tandem duplication of MLL should not be placed in this category.
d) Three new cytogenetically defined entities are added: AML with t(6;9)(p23;q23); DEK-NUP214, AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1; and AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1.
e) Two provisional entities are added: AML with mutated NPM1 and AML with mutated CEBPA. Although not included as a distinct entity, examination for mutations of FLT3 is strongly recommended in all cytogenetically normal AML.

2. AML with myelodysplasia-related changes.
a) Name changed from "AML with multilineage dysplasia".
b) Cases of AML are assigned to this category if 1) they have a previous history of MDS and have evolved to AML, 2) they have a myelodysplasia-related cytogenetic abnormality, or 3) if ≥50% of cells in two or more myeloid lineages are dysplastic.
3. Therapy-related myeloid neoplasms.
Cases are no longer subcategorized as "alkylating agent related" or "topoisomerase II-inhibitor related" or "other".

4. AML, NOS.
a) Some cases previously assigned to the subcategory of AML, NOS as acute erythroid leukaemia may be re-classified as AML with myelodysplasia-related changes.
b) Cases previously categorized as AML, NOS, acute megakaryoblastic leukaemia should be placed in the appropriate ­genetic category if they are associated with inv(3)(q21q26.2) or t(3;3)(q21;q26.2); RPN1-EVI1, or AML (megakaryoblastic) with t(1;22)(p13;q13); RBM15-MKL1. Down syndrome related cases are excluded from this category as well.

5. Myeloid proliferations related to Down syndrome.
New category to incorporate transient abnormal myelopoiesis associated with Down syndrome as well as myeloid leukaemia associated with Down syndrome . MDS related to Down syndrome is considered biologically identical to AML ­related to Down syndrome.







James W. Vardiman
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James W. Vardiman
Department of Pathology
University of Chicago Medical Center
Chicago
USA




Richard D. Brunning
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Richard D. Brunning
Department of Laboratory Medicine and Pathology
University of Minnesota
Minneapolis
USA




Daniel A. Arber
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Daniel A. Arber
Stanford University Medical Center
Stanford
USA




Michelle M. Le Beau
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Michelle M. Le Beau
Cancer Cytogenetics Laboratory
University of Chicago
Chicago
USA




Anna Porwit
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Anna Porwit
Department of Pathology
Karolinska University Hospital
Stockholm
Sweden




Ayalew Tefferi
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Ayalew Tefferi
Division of Hematology
Mayo Clinic
Rochester
USA




Clara D. Bloomfield
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Clara D. Bloomfield
The Ohio State University
519 James Cancer Hospital
Columbus
USA




Jürgen Thiele
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Jürgen Thiele
Institute of Pathology
University of Cologne
Cologne
Germany





Adequate bone marrow trephine biopsy
Adequate bone marrow trephine biopsy

MDS in well-processed bone marrow biopsy
MDS in well-processed bone marrow biopsy

AML.  Agranular myeloblasts
AML. Agranular myeloblasts

AML Granulated myeloblasts.
AML Granulated myeloblasts.

Monoblasts, promonocytes and abnormal monocytes
Monoblasts, promonocytes and abnormal monocytes

Antigen expression during myeloid differentiation
Antigen expression during myeloid differentiation

Tyrosine kinase genes often affected in MPN
Tyrosine kinase genes often affected in MPN

JAK2 signaling pathway
JAK2 signaling pathway

Mutations in AML pathogenesis
Mutations in AML pathogenesis