The World Health Organization (WHO) classification of tumors of the hematopoietic and lymphoid tissues was last updated in 2008. Since then, there have been numerous advances in the identification of unique biomarkers associated with some myeloid neoplasms and acute leukemias, largely derived from gene expression analysis and next-generation sequencing that can significantly improve the diagnostic criteria as well as the prognostic relevance of entities currently included in the WHO classification and that also suggest new entities that should be added. Therefore, there is a clear need for a revision to the current classification. The revisions to the categories of myeloid neoplasms and acute leukemia will be published in a monograph in 2016 and reflect a consensus of opinion of hematopathologists, hematologists, oncologists, and geneticists. The 2016 edition represents a revision of the prior classification rather than an entirely new classification and attempts to incorporate new clinical, prognostic, morphologic, immunophenotypic, and genetic data that have emerged since the last edition. The major changes in the classification and their rationale are presented here.

For these reasons, the fourth edition is being updated, but this 2016 classification is not a major overhaul of the disease categories. Rather, it is intended to incorporate new knowledge of these disorders obtained since the 2008 publication and is a revision of that classification. The purpose of this report is to summarize the major changes in the revised WHO classification of myeloid neoplasms and acute leukemia and to provide the rationale for those changes. Table 1 lists the major subtypes of myeloid neoplasms and acute leukemias according to the updated (2016) WHO classification.

In collaboration with the Society for Hematopathology and the European Association for Haematopathology, the World Health Organization (WHO) published the third and fourth editions of the WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, in 2001 and 2008, respectively, as part of a series of WHO Classification of Tumours “blue book” monographs. In the spring of 2014, a clinical advisory committee (CAC) composed of ∼100 pathologists, hematologists, oncologists, and geneticists from around the world convened to propose revisions to the fourth edition of the classification. The revision of the fourth edition follows the philosophy of the third and fourth editions to incorporate clinical features, morphology, immunophenotyping, cytogenetics, and molecular genetics to define disease entities of clinical significance. The fourth edition of the classification of hematopoietic and lymphoid tissues was the second volume of the WHO “blue book” tumor series, and the series publication is still in progress. A fifth edition series cannot begin until the fourth edition series is completed; but after 8 years of information and experience that have emerged from scientific and clinical studies, a revision of these criteria for hematopoietic and lymphoid neoplasms was felt to be necessary and timely. In relation to myeloid neoplasms and acute leukemia, this revision has been influenced by several factors including the following:

The revised criteria for CNL, PV, ET, PMF, and prePMF are listed in Tables 3 - 7 in addition to a slightly modified grading of reticulin and collagen BM fibers ( Table 8 ). It is important to emphasize that an accurate histologic diagnosis has been proven to be key to predict prognosis in this group of diseases. 13

With regard to chronic myeloid leukemia (CML), BCR-ABL1 + , most cases of CML in chronic phase can be diagnosed from peripheral blood (PB) findings combined with detection of t(9;22)(q34.1;q11.2) or, more specifically, BCR-ABL1 by molecular genetic techniques. However, a bone marrow (BM) aspirate is essential to ensure sufficient material for a complete karyotype and for morphologic evaluation to confirm the phase of disease. 1 , 2 In the era of tyrosine-kinase inhibitor (TKI) therapy, newly diagnosed patients may have a nearly normal lifespan, but regular monitoring for BCR-ABL1 burden and for evidence of genetic evolution and development of resistance to TKI therapy is essential to detect disease progression. 3 , 4 Although the accelerated phase (AP) of CML is becoming less common in the era of TKI therapy, there are no universally accepted criteria for its definition. The criteria for AP in the revised WHO classification include hematologic, morphologic, and cytogenetic parameters which are supplemented by additional parameters usually attributed to genetic evolution, 5 and manifested by evidence of resistance to TKIs (see Table 2 ). These latter “response to TKI therapy” criteria for AP are considered as “provisional” until further supported by additional data. Diagnosis of blast phase (BP) still requires either at least 20% blasts in the blood or BM or the presence of an extramedullary accumulation of blasts. However, because the onset of lymphoid BP may be quite sudden, the detection of any bona fide lymphoblasts in the blood or marrow should raise concern for a possible impending lymphoid BP, and prompt additional laboratory and genetic studies to exclude this possibility.

The categories of MPNs have not significantly changed since the 2008 fourth edition of the classification, but discoveries of new mutations and improved understanding of the morphologic features of some entities have impacted the diagnostic criteria for the disease entities. Mastocytosis, however, is no longer considered a subgroup of the MPNs due to its unique clinical and pathologic features, ranging from indolent cutaneous disease to aggressive systemic disease, and is now a separate disease category in the classification.

As mentioned, mastocytosis is no longer listed under the broad heading of MPNs. Major advances in the understanding of mastocytosis have been made since the 2008 classification, 19 and these are incorporated into the text of the monograph. Table 9 20 lists the 2016 categories of mastocytosis, which includes a shortening of the name of the 2008 category of “systemic mastocytosis with associated clonal hematological non-mast-cell lineage disease (SH-AHNMD)” to the 2016 category of “systemic mastocytosis with an associated hematological neoplasm (SM-AHN).” In many cases, the AHN is an aggressive neoplasm that must be treated and the diagnosis should clearly and separately indicate the presence of this disorder in a distinct diagnosis line.

The criteria for the diagnosis of the eosinophilia-related proliferations associated with specific molecular genetic changes are retained in the classification, although it is noted that eosinophilia may be absent in a subset cases. In the 2016 revision ( Table 10 ), this disease group will incorporate the myeloid neoplasm with t(8;9)(p22;p24.1);PCM1-JAK2 as a new provisional entity. 21 , 22 This rare entity is characterized by a combination of eosinophilia with BM findings of left-shifted erythroid predominance, lymphoid aggregates, and often myelofibrosis, at times mimicking PMF. It can also rarely present as T- or B-lymphoblastic leukemia (acute lymphoblastic leukemia [ALL]) and responds to JAK inhibition. 23 Other JAK2-rearranged neoplasms, for example, t(9;12)(p24.1;p13.2);ETV6-JAK2 and t(9;22)(p24.1;q11.2);BCR-JAK2 may have similar features, but are uncommon and are not currently included as distinct entities. Moreover, ETV6-JAK2 and BCR-JAK2–rearranged neoplasms present primarily as B-cell ALL (B-ALL), and these are best considered as BCR-ABL1–like B-ALL, a new provisional category of B-lymphoblastic leukemia/lymphoma. 22

In MDS/MPN, the karyotype is often normal or shows abnormalities in common with MDS. Targeted sequencing of genes mutated in myeloid neoplasms detects mutations in a high proportion of cases of chronic myelomonocytic leukemia (CMML) as well as other MDS/MPN patients. 26 The most commonly mutated genes in CMML are SRSF2, TET2, and/or ASXL1 (>80% of cases). 26 , 27 Other mutations which occur at lower frequency include SETBP1, NRAS/KRAS, RUNX1, CBL, and EZH2. 28 , 29 They can be helpful adjunct studies in difficult cases, particularly given the frequently normal karyotype of CMML, but should not be used alone as proof of neoplasia because some of these mutations occur in healthy older patients as so-called clonal hematopoiesis of indeterminate potential (CHIP) 30 , 31 (for further discussion, see “Myelodysplastic syndromes”). ASXL1 is a predictor of aggressive disease behavior and has been incorporated into a prognostic scoring system for CMML alongside karyotype and clinicopathologic parameters. 27 Of note, NPM1 mutation is seen in a rare subset of CMML (3%-5%) and appears also to herald a more aggressive clinical course.

The myelodysplastic syndrome (MDS)/MPN category was introduced in the third edition to include myeloid neoplasms with clinical, laboratory, and morphologic features that overlap between MDS and MPN. 24 Based on accumulated scientific evidence, a provisional entity within the MDS/MPN unclassifiable group, refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T), has been accepted as a full entity, now termed MDS/MPN with ring sideroblasts and thrombocytosis in the 2016 revision. The 2016 revised criteria for diseases in this category are summarized in Tables 11 - 14 . 25

A diagnosis of CMML requires both the presence of persistent PB monocytosis ≥1 × 10 9 /L and monocytes accounting for ≥10% of the white blood cell (WBC) differential count. Due to the discovery of molecular and clinical differences between the so-called “proliferative type” of CMML (WBC count ≥13 × 10 9 /L) and the “dysplastic type” (WBC <13 × 10 9 /L), particularly those differences related to aberrancies in the RAS/MAPK signaling pathways, 32-34 the separation of CMML into these subtypes is warranted. In addition, blast percentage maintains clear prognostic importance in CMML as initially suggested in the third edition and later confirmed in the fourth edition. Recent evidence has shown that a more precise prognostication can be obtained with 3 blast-based groupings: CMML-0, a category for cases with <2% blasts in PB and <5% blasts in BM; CMML-1 for cases with 2% to 4% blasts in PB and/or 5% to 9% blasts in BM; and CMML-2 for cases with 5% to 19% blasts in PB, 10% to 19% in BM, and/or when any Auer rods are present. 33 , 35 The revision incorporates the CMML-0 category into the classification scheme. In view of the importance of separating promonocytes (blast equivalent cells) from monocytes, which can have abnormal features in CMML, precise morphologic evaluation is essential, with the appropriate integration of flow cytometry immunophenotyping and cytogenetic and molecular genetic testing. Because other disorders must be excluded before a diagnosis of CMML can be made, BCR-ABL1 rearrangement should be excluded in all cases and PDGFRA, PDGFRB, FGFR1 rearrangements or PCM1-JAK2 fusions excluded if eosinophilia is present. A prior well-documented diagnosis of a MPN would also generally exclude CMML or another type of MDS/MPN. 36 , 37

The rare MDS/MPN subtype atypical CML (aCML) is now better characterized molecularly and can be more easily separated from CNL, a rare subtype of MPN similarly characterized by neutrophilia. Although CNL is strongly associated with the presence of CSF3R mutations, these appear to be very rare in aCML (<10%). 38 Conversely, aCML is associated with SETBP1 and/or ETNK1 mutations in up to a third of cases. 28 , 39 , 40 The so-called MPN-associated driver mutations (JAK2, CALR, MPL) are typically absent in aCML.

The criteria for MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T; previously known as RARS-T) include thrombocytosis (≥450 × 10 9 /L) associated with refractory anemia, dyserythropoiesis in the BM with ring sideroblasts accounting for 15% or more of erythroid precursors, and megakaryocytes with features resembling those in PMF or ET. After the discovery that MDS/MPN-RS-T is frequently associated with mutations in the spliceosome gene SF3B1 (which in turn are associated with the presence of ring sideroblasts), there is now enough evidence to support MDS/MPN-RS-T as a full entity. 41-44 In MDS/MPN-RS-T, SF3B1 is often comutated with JAK2 V617F or less frequently (<10%) with CALR, or MPL genes, thus providing a biological explanation for the true hybrid nature of this rare myeloid neoplasm. Unlike MDS with ring sideroblasts (see “Myelodysplastic syndromes”), the number of ring sideroblasts required for a diagnosis of MDS/MPN-RS-T is not altered by the presence or absence of a mutation in SF3B1. Because of changes in the MDS terminology (see “Myelodysplastic syndromes”), the name RARS-T was changed to MDS/MPN-RS-T.

Juvenile myelomonocytic leukemia (JMML) is an aggressive clonal hematopoietic disorder of infancy and early childhood characterized by an excessive proliferation of cells of monocytic and granulocytic lineages that is included as a MDS/MPN subtype. 45 , 46 Approximately 90% of patients carry either somatic or germ line mutations of PTPN11, KRAS, NRAS, CBL, or NF1. These genetic aberrations are largely mutually exclusive and activate the RAS/MAPK pathway. The clinical and pathological findings of JMML are not substantially changed from the current WHO fourth edition (2008). However, molecular diagnostic parameters have been refined. The updated diagnostic findings are listed in Table 14 .

The MDS are a group of clonal BM neoplasms characterized by ineffective hematopoiesis, manifested by morphologic dysplasia in hematopoietic cells and by peripheral cytopenia(s). The revised classification introduces refinements in morphologic interpretation and cytopenia assessment and addresses the influence of rapidly accumulating genetic information in MDS diagnosis and classification. Cytopenia is a “sine qua non” for any MDS diagnosis and in prior classifications, MDS nomenclature included references to “cytopenia” or to specific types of cytopenia (eg, “refractory anemia”). However, the WHO classification relies mainly on the degree of dysplasia and blast percentages for disease classification and specific cytopenias have only minor impact on MDS classification. Moreover, the lineage(s) manifesting significant morphologic dysplasia frequently do not correlate with the specific cytopenia(s) in individual MDS cases.47-49 For these reasons, the terminology for adult MDS has changed to remove terms such as “refractory anemia” and “refractory cytopenia” and replaces them with “myelodysplastic syndrome” followed by the appropriate modifiers: single vs multilineage dysplasia, ring sideroblasts, excess blasts, or the del(5q) cytogenetic abnormality (see Table 15). There are no changes to childhood MDS; refractory cytopenia of childhood remains as a provisional entity within this category.

Table 15 Name . Dysplastic lineages . Cytopenias* . Ring sideroblasts as % of marrow erythroid elements . BM and PB blasts . Cytogenetics by conventional karyotype analysis . MDS with single lineage dysplasia (MDS-SLD) 1 1 or 2 <15%/<5%† BM <5%, PB <1%, no Auer rods Any, unless fulfills all criteria for MDS with isolated del(5q) MDS with multilineage dysplasia (MDS-MLD) 2 or 3 1-3 <15%/<5%† BM <5%, PB <1%, no Auer rods Any, unless fulfills all criteria for MDS with isolated del(5q) MDS with ring sideroblasts (MDS-RS) MDS-RS with single lineage dysplasia (MDS-RS-SLD) 1 1 or 2 ≥15%/≥5%† BM <5%, PB <1%, no Auer rods Any, unless fulfills all criteria for MDS with isolated del(5q) MDS-RS with multilineage dysplasia (MDS-RS-MLD) 2 or 3 1-3 ≥15%/≥5%† BM <5%, PB <1%, no Auer rods Any, unless fulfills all criteria for MDS with isolated del(5q) MDS with isolated del(5q) 1-3 1-2 None or any BM <5%, PB <1%, no Auer rods del(5q) alone or with 1 additional abnormality except −7 or del(7q) MDS with excess blasts (MDS-EB) MDS-EB-1 0-3 1-3 None or any BM 5%-9% or PB 2%-4%, no Auer rods Any MDS-EB-2 0-3 1-3 None or any BM 10%-19% or PB 5%-19% or Auer rods Any MDS, unclassifiable (MDS-U) with 1% blood blasts 1-3 1-3 None or any BM <5%, PB = 1%,‡ no Auer rods Any with single lineage dysplasia and pancytopenia 1 3 None or any BM <5%, PB <1%, no Auer rods Any based on defining cytogenetic abnormality 0 1-3 <15%§ BM <5%, PB <1%, no Auer rods MDS-defining abnormality Refractory cytopenia of childhood 1-3 1-3 None BM <5%, PB <2% Any Name . Dysplastic lineages . Cytopenias* . Ring sideroblasts as % of marrow erythroid elements . BM and PB blasts . Cytogenetics by conventional karyotype analysis . MDS with single lineage dysplasia (MDS-SLD) 1 1 or 2 <15%/<5%† BM <5%, PB <1%, no Auer rods Any, unless fulfills all criteria for MDS with isolated del(5q) MDS with multilineage dysplasia (MDS-MLD) 2 or 3 1-3 <15%/<5%† BM <5%, PB <1%, no Auer rods Any, unless fulfills all criteria for MDS with isolated del(5q) MDS with ring sideroblasts (MDS-RS) MDS-RS with single lineage dysplasia (MDS-RS-SLD) 1 1 or 2 ≥15%/≥5%† BM <5%, PB <1%, no Auer rods Any, unless fulfills all criteria for MDS with isolated del(5q) MDS-RS with multilineage dysplasia (MDS-RS-MLD) 2 or 3 1-3 ≥15%/≥5%† BM <5%, PB <1%, no Auer rods Any, unless fulfills all criteria for MDS with isolated del(5q) MDS with isolated del(5q) 1-3 1-2 None or any BM <5%, PB <1%, no Auer rods del(5q) alone or with 1 additional abnormality except −7 or del(7q) MDS with excess blasts (MDS-EB) MDS-EB-1 0-3 1-3 None or any BM 5%-9% or PB 2%-4%, no Auer rods Any MDS-EB-2 0-3 1-3 None or any BM 10%-19% or PB 5%-19% or Auer rods Any MDS, unclassifiable (MDS-U) with 1% blood blasts 1-3 1-3 None or any BM <5%, PB = 1%,‡ no Auer rods Any with single lineage dysplasia and pancytopenia 1 3 None or any BM <5%, PB <1%, no Auer rods Any based on defining cytogenetic abnormality 0 1-3 <15%§ BM <5%, PB <1%, no Auer rods MDS-defining abnormality Refractory cytopenia of childhood 1-3 1-3 None BM <5%, PB <2% Any

One of the biggest challenges in this category is separating MDS from reactive causes of cytopenia and dysplasia. Although the threshold to define dysplasia will remain as 10% dysplastic cells in any hematopoietic lineage, it is recognized that dysplasia in excess of 10% may occur in some normal individuals and even more frequently in nonneoplastic causes of cytopenia.50,51 Moreover, identification of dysplasia is not always reproducible among even experienced hematopathologists.52,53 For these reasons, possible reactive etiologies of dysplasia should always be carefully considered prior to making a diagnosis of MDS, particularly when the dysplasia is subtle and limited to 1 lineage. Some dysplastic changes, particularly the presence of micromegakaryocytes (which can be highlighted by immunostaining for megakaryocyte markers in the BM trephine), are relatively specific for myelodysplasia and have high reproducibility.53

The myeloblast percentage, as determined by counting well-prepared, cellular BM aspirate smears and/or touch preparations and a PB smear, remains critical in defining the WHO MDS categories and as risk strata in the Revised International Prognostic Scoring System (IPSS-R).54 The presence of 1% blasts in the PB, with <5% BM blasts, defines 1 type of MDS, unclassifiable (MDS-U). However, because 1% blasts may not be reproducible as a single observation, this finding must now be demonstrated on at least 2 separate occasions in order to diagnose MDS-U according to this criterion. There is a major change in the diagnostic criteria for myeloid neoplasms with erythroid predominance (erythroid precursors ≥50% of all BM cells). In the updated classification, the denominator used for calculating blast percentage in all myeloid neoplasms is all nucleated BM cells, not just the “nonerythroid cells.” This will result in most cases previously diagnosed as the erythroid/myeloid subtype of acute erythroid leukemia now being classified as MDS with excess blasts, as discussed in “AML, not otherwise specified” (see Table 16).

Table 16 BM erythroid precursors . Myeloblast % of all cells in BM (or PB) . Prior therapy? . Recurring WHO genetic abnormality? . Meets criteria for AML-MRC? . Fourth edition diagnosis . Updated fourth edition diagnosis . ≥50% NA Yes NA NA Therapy-related myeloid neoplasm Therapy-related myeloid neoplasm ≥50% ≥20% No Yes NA AML with recurring genetic abnormality AML with recurring genetic abnormality ≥50% ≥20% No No Yes AML with myelodysplasia-related changes AML with myelodysplasia-related changes ≥50% ≥20% No No No AML, NOS, acute erythroid leukemia (erythroid/myeloid type) AML, NOS (non erythroid subtype) ≥50% <20%, but ≥20% of nonerythroid cells No No* NA AML, NOS, acute erythroid leukemia (erythroid/myeloid subtype) MDS† ≥50% <20%, and <20% of nonerythroid cells No No* NA MDS† MDS† >80% immature erythroid precursors with ≥30% proerythroblasts <20% No No* NA AML, NOS, acute erythroid leukemia (pure erythroid type) AML, NOS, acute erythroid leukemia (pure erythroid type) BM erythroid precursors . Myeloblast % of all cells in BM (or PB) . Prior therapy? . Recurring WHO genetic abnormality? . Meets criteria for AML-MRC? . Fourth edition diagnosis . Updated fourth edition diagnosis . ≥50% NA Yes NA NA Therapy-related myeloid neoplasm Therapy-related myeloid neoplasm ≥50% ≥20% No Yes NA AML with recurring genetic abnormality AML with recurring genetic abnormality ≥50% ≥20% No No Yes AML with myelodysplasia-related changes AML with myelodysplasia-related changes ≥50% ≥20% No No No AML, NOS, acute erythroid leukemia (erythroid/myeloid type) AML, NOS (non erythroid subtype) ≥50% <20%, but ≥20% of nonerythroid cells No No* NA AML, NOS, acute erythroid leukemia (erythroid/myeloid subtype) MDS† ≥50% <20%, and <20% of nonerythroid cells No No* NA MDS† MDS† >80% immature erythroid precursors with ≥30% proerythroblasts <20% No No* NA AML, NOS, acute erythroid leukemia (pure erythroid type) AML, NOS, acute erythroid leukemia (pure erythroid type)

Despite the lowering of the neutropenia prognostic threshold in the IPSS-R to 0.8 × 109/L,54 the WHO thresholds defining cytopenia will remain as in the original IPSS (hemoglobin, <10 g/dL; platelets, <100 × 109/L; absolute neutrophil count, <1.8 × 109/L); a diagnosis of MDS may be made in rare cases with milder levels of cytopenia, but at least 1 cytopenia must be present in order to make the diagnosis. It should be noted that some ethnic groups may have a reference range for normal absolute neutrophil count that is lower than 1.8 × 109/L, and thus caution should be exercised in interpreting neutropenia if it is the only cytopenia. MDS-U will continue to include cases with single lineage dysplasia or isolated del(5q) and pancytopenia, but in such cases all PB counts must be below the WHO thresholds given in this paragraph.

The same cytogenetic abnormalities listed in the 2008 WHO classification55 remain MDS-defining in a cytopenic patient, even in the absence of diagnostic morphologic dysplasia. In such cases, the abnormality must be demonstrated by conventional karyotyping, not by fluorescence in situ hybridization (FISH) or sequencing technologies. The presence of +8, −Y, or del(20q) is not considered to be MDS-defining in the absence of diagnostic morphologic features of MDS. In spite of the increased knowledge of the prognostic importance of genetic findings in MDS, del(5q) remains as the only cytogenetic or molecular genetic abnormality that defines a specific MDS subtype. Based on recent data showing no adverse effect of 1 chromosomal abnormality in addition to the del(5q),56-58 the entity MDS with isolated del(5q) may be diagnosed if there is 1 additional cytogenetic abnormality besides the del(5q), unless that abnormality is monosomy 7 or del(7q). Even though cytogenetic findings are not used to define other specific subtypes of MDS, they are strongly correlated with prognosis, as reflected in the 5 cytogenetic prognostic groups in the IPSS-R scheme54,58 ; thus, a complete BM karyotype remains a critical test in any newly diagnosed MDS case.

As with all the other myeloid neoplasms, a large amount of data has recently become available on recurring mutations in MDS. Targeted sequencing of a limited number of genes can detect mutations in 80% to 90% of MDS patients; the most commonly mutated genes in MDS are SF3B1, TET2, SRSF2, ASXL1, DNMT3A, RUNX1, U2AF1, TP53, and EZH2.59,60 Importantly, acquired clonal mutations identical to those seen in MDS can occur in the hematopoietic cells of apparently healthy older individuals without MDS, so-called “clonal hematopoiesis of indeterminate potential” (CHIP).30,31,61 Although some patients with CHIP subsequently develop MDS, the natural history of this condition is not yet fully understood; thus, the presence of MDS-associated somatic mutations alone is not considered diagnostic of MDS in this classification, even in a patient with unexplained cytopenia, where these mutations may be commonly found.62 Further study is required to determine the optimal management and monitoring of such patients and to investigate possible links between specific mutations, mutant allele fraction, or mutation combinations and subsequent development of bona fide MDS.63 Rare cases of familial MDS are associated with germ line mutations, which can be investigated by sequencing non-MDS patient tissue.

The number and types of specific mutations are strongly associated with disease outcome in MDS, and the addition of mutation data improves the prognostic value of existing risk-stratification schemes in MDS.64,65 TP53 mutation is associated with aggressive disease in MDS in general66 and appears to predict poorer response to lenalidomide in patients with del(5q).67-69 Evaluation for TP53 mutation is recommended in patients with MDS with isolated del(5q) to help identify an adverse prognostic subgroup in this generally favorable prognosis MDS entity.