Cytoplasmic nucleophosmin is not detected in blastic plasmacytoid dendritic cell neoplasm

Acute myeloid leukemia carrying cytoplasmic mutated nucleophosmin (NPMc⁺ AML) and blastic plasmacytoid dendritic cell neoplasm have been included as new entities in the 4^th edition (2008) WHO classification of myeloid neoplasms. These conditions may show clinical and pathological overlapping features (leukemic and skin involvement, and expression of macrophage markers). In this study, we provide evidence that aberrant cytoplasmic dislocation of nucleophosmin – the immunohistochemical surrogate for NPM1 mutations – allows the two entities to be genetically separated. In fact, nucleophosmin is consistently cytoplasmic in NPMc⁺ AML (because of the presence of NPM1 mutations), whilst it is nucleus-restricted (predictive of a germline NPM1 gene) in blastic plasmacytoid dendritic cell neoplasm. Our results clearly point cytoplasmic nucleophosmin (a full predictor of NPM1 mutations) as a new marker for distinguishing NPMc⁺ AML and blastic plasmacytoid dendritic cell neoplasm, further clarify the cell of origin of NPMc⁺ AML, and justify the inclusion of these pathological conditions as separate entities in the new WHO classification.     

Diagnostic work‐up of acute myeloid leukemia

Acute myeloid leukemia (AML) is characterized by a clonal expansion of undifferentiated myeloid precursors resulting in impaired hematopoiesis and bone marrow failure. In 2016, the World Health Organization (WHO) published revisions to the classification of myeloid neoplasms and acute leukemias. Similar to the 2008 classification, the updated classification incorporates clinical features, morphology, immunophenotyping, and cytogenetics, with greater emphasis on molecular genetics, to define disease entities. This brief review addresses the various components of pathologic assessment to establish a diagnosis of AML and to help risk stratify patients, with an emphasis on newer techniques used in the detection of mutations with prognostic significance, as well as assays employed in the evaluation of minimal residual disease following treatment.     

Molecular markers in acute myeloid leukaemia

An increasing number of cytogenetic and molecular genetic aberrations have been identified in acute myeloid leukaemia (AML), highlighting the biological heterogeneity of the disease. Moreover, the characterisation of specific molecular abnormalities provides the basis for targeted therapies, such as all trans retinoic acid (ATRA) and arsenic trioxide treatment in acute promyelocytic leukaemia or tyrosine kinase inhibitors in AML with FLT3 mutations. Several cytogenetic and molecular genetic changes have been shown to be prognostically relevant and have been acknowledged in the latest WHO classification of AML as separate entities. A detailed marker assessment at diagnosis is crucial for risk-stratification of AML patients, allowing the identification of those at high risk of relapse, who may benefit from early allogeneic stem cell transplantation. Finally, molecular markers are important for the detection of minimal residual disease after initial therapy and during long-term follow-up, which enables a more tailored treatment approach for individual AML patients.     

Controversies in the recent (2016) World Health Organization classification of acute myeloid leukemia

The current World Health Organization (WHO) Classification of acute myeloid leukemia (AML), developed in 2016 and published in 2017, codifies the defining features of AML and recognizes several subtypes based on clinical, morphologic, and genetic features. This classification is widely used for the purposes of assigning patients to specific therapeutic approaches and entry into clinical trials. Although the WHO Classification ultimately has its origins in the original 1976 French-American-British Classification, it has been periodically updated by the incorporation of a large body of evidence and input from both diagnosticians and clinicians who study and treat AML. Nevertheless, the recent accumulation of genetic data on the molecular underpinnings of myeloid neoplasms as well as numerous recently approved novel therapies have highlighted areas of controversy in how we currently define and classify AML; the 2016 WHO Classification will continually be revised and updated in future versions based on these advances. The purpose of this review is to explore areas of potential refinement in the current WHO Classification of AML, both in terms of its criteria defining the disease as well as the specific disease subtypes.     

Clinical implications of molecular markers in acute myeloid leukemia

The recently updated World Health Organization (WHO) Classification of myeloid neoplasms and leukemia reflects the fact that research in the underlying pathogenic mechanisms of acute myeloid leukemia (AML) has led to remarkable advances in our understanding of the disease. Gene mutations now allow us to explore the enormous diversity among cytogenetically defined subsets of AML, particularly the large subset of cytogenetically normal AML. Despite the progress in unraveling the tumor genome, only a small number of recurrent mutations have been incorporated into risk‐stratification schemes and have been proven to be clinically relevant, targetable lesions. We here discuss the utility of molecular markers in AML in prognostication and treatment decision making, specifically highlighting the aberrations included in the current WHO classification.     

Diagnosis of acute myeloid leukemia according to the WHO classification in the Japan Adult Leukemia Study Group AML-97 protocol

We reviewed and categorized 638 of 809 patients who were registered in the Japan Adult Leukemia Study Group acute myeloid leukemia (AML)-97 protocol using morphological means. Patients with the M3 subtype were excluded from the study group. According to the WHO classification, 171 patients (26.8%) had AML with recurrent genetic abnormalities, 133 (20.8%) had AML with multilineage dysplasia (MLD), 331 (51.9%) had AML not otherwise categorized, and 3 (0.5%) had acute leukemia of ambiguous lineage. The platelet count was higher and the rate of myeloperoxidase (MPO)-positive blasts was lower in AML with MLD than in the other WHO categories. The outcome was significantly better in patients with high (≥50%) than with low (<50%) ratios of MPO-positive blasts (P < 0.01). The 5-year survival rates for patients with favorable, intermediate, and adverse karyotypes were 63.4, 39.1, and 0.0%, respectively, and 35.5% for those with 11q23 abnormalities (P < 0.0001). Overall survival (OS) did not significantly differ between nine patients with t(9;11) and 23 with other 11q23 abnormalities (P = 0.22). Our results confirmed that the cytogenetic profile, MLD phenotype, and MPO-positivity of blasts are associated with survival in patients with AML, and showed that each category had the characteristics of the WHO classification such as incidence, clinical features, and OS.     

Prospective study of 174 de novo acute myelogenous leukemias according to the WHO classification: subtypes, cytogenetic features and FLT3 mutations

We report a prospective study of 174 unselected adult de novo acute myeloid leukemia (AML) cases diagnosed using the WHO classification. Of those, 57 (33%) were AML with recurrent cytogenetic abnormalities, 41 were (24%) AML with multilineage dysplasia, 74 (42%) were AML not otherwise categorized, and two were acute leukemias of ambiguous lineage. Clonal cytogenetic abnormalities were detected in 64% of the WHO AML cases with t(15;17) (15%), t(8;21) (12%), +8 (11%), -7/del7q (8%) and del9q (5%) being the most common ones. The FLT3/ITD mutations (FMS-like tyrosine kinase 3/internal tandem duplication) were observed in 12% of the WHO AML cases, which is much lower than ones in the literature, while the 6% incidence of the FLT3-activating loop mutations (either FLT3/D835 or FLT3/I836) was comparable with others. Both mutations were associated with leukocytosis. Our study also suggests that the FLT3 mutations are biomarkers independent of cytogenetic characteristics.     

MLD according to the WHO classification in AML has no correlation with age and no independent prognostic relevance as analyzed in 1766 patients

Between February 1996 and December 2004, the German Leukemia Study Initiative registered 1766 consecutive patients for the acute myeloid leukemia (AML) 96 study, all of whom were diagnosed by central cytomorphology according to the French-American-British (FAB) and the new World Health Organization (WHO) classification. We focused our analysis on the prognostic impact of multilineage dysplasia (MLD) as a new parameter of the WHO classification for AML. We could not confirm the WHO statement that MLD occurs most frequently in older individuals, but we confirmed that MLD is often associated with an unfavorable cytogenetic profile (P < .001). In 1332 individuals receiving intensive AML therapy presence of MLD was negatively correlated with complete remission (P = .001) in univariate, but not in multivariate, analysis. Multivariate analysis of either event-free or overall survival again failed to show an independent prognostic significance of MLD besides age, cytogenetics, and, in part, NPM1/FLT3-ITD mutations. Our data support a reassessment of the WHO classification in the light of a more biologic understanding of AML. This study is registered at www.ClinicalTrials.gov as #NCT00180115.     

Akute myeloische Leukämie

Acute myeloid leukemia (AML) is a genetically heterogeneous disease. The genetic diagnostics have become an essential component in the initial work-up for disease classification, prognostication and prediction. More and more promising molecular targeted therapeutics are becoming available. A prerequisite for individualized treatment strategies is a fast pretherapeutic molecular screening including the fusion genes PML-RARA, RUNX1-RUNX1T1 and CBFB-MYH11 as well as mutations in the genes NPM1, FLT3 and CEBPA. Promising new therapeutic approaches include the combination of all- trans retinoic acid and arsentrioxid in acute promyelocytic leukemia, the combination of intensive chemotherapy with KIT inhibitors in core-binding factor AML and FLT3 inhibitors in AML with FLT3 mutation, as well as gemtuzumab ozogamicin therapy in patients with low and intermediate cytogenetic risk profiles. With the advent of the next generation sequencing technologies it is expected that new therapeutic targets will be identified. These insights will lead to a further individualization of AML therapy.     

Characteristics of acute myeloid leukemia with myelodysplasia‐related changes: A retrospective analysis in a cohort of Chinese patients

We retrospectively analyzed 449 patients with AML under the WHO classification of AML 2008 and probed implications of this classification in diagnosis and treatment of acute myeloid leukemia with myelodysplasia‐related changes (AML‐MRC) among them. The clinical presentations, biological features, treatments, and prognosis of patients diagnosed with AML‐MRC were analyzed and compared with those of AML not otherwise specified (AML‐NOS). In all patients, 115 (25.6%) were diagnosed as AML‐MRC including 64 males and 51 females with median onset age of 48 years (range from 17 to 78). Their complete remission (CR) rate was 60.9% and relapse rate was 57.1%. The observed median overall survival (OS) and disease‐free survival (DFS) were 10 and 5 months, respectively, which was significantly shorter than those of AML‐NOS patients (P < 0.05). The prognosis of AML‐MRC patients with myelodysplastic syndrome (MDS)‐related cytogenetics sole was similar to those with history of MDS or myelodysplastic/myeloproliferative neoplasm (MDS/MPN). Patients with MDS‐related cytogenetic abnormalities and/or history of MDS or MDS/MPN predisposed significantly shortened CR, OS, and DFS than AML‐MRC patients with only multilineage dysplasia (MLD) and AML‐NOS patients (P < 0.05). Multivariate analysis showed that age, cytogenetics, and history of MDS or MDS/MPN were independent prognostic factors. Patient diagnosed as AML‐MRC presented distinctive clinical and biological features. Presence of MLD does not change the prognosis. Am. J. Hematol. 89:874–881, 2014. © 2014 Wiley Periodicals, Inc.     

Familial CEBPA-mutated acute myeloid leukemia

Familial CEBPA-mutated acute myeloid leukemia (AML) represents a recognized leukemia predisposition syndrome, with several families described in the literature since the initial report in 2004. The pathological features and long-term survival of individuals with familial CEBPA-mutated AML are reminiscent of sporadic CEBPAdm AML.  Germline mutations predominantly localize to the N-terminal and are associated with near complete penetrance, with age of AML onset from 2–50 years, frequently accompanied by the acquisition of a second CEBPA mutation in C-terminal domain.  Patients appear to have a significant risk of late AML recurrence and these typically represent independent leukemic episodes, characterized by a unique molecular profile that is distinct from that of the preceding tumor.  While these patients respond well to salvage therapies, allogeneic hematopoietic stem cell transplantation (HSCT) should be considered for patients with high-risk features at presentation or recurrent disease, with the aim of eradicating the germline mutation and improving long-term survival. In contrast, inherited C-terminal CEBPA mutations occur less frequently and appear to demonstrate reduced penetrance, impeding clinical detection and surveillance.     

Mutated nucleophosmin detects clonal multilineage involvement in acute myeloid leukemia: Impact on WHO classification

Because of a lack of specific clonality markers, information on lineage involvement and cell of origin of acute myeloid leukemia with normal karyotype (AML-NK), is missing. Because Nucleophosmin (NPM) gene is frequently mutated in AML-NK and causes aberrant NPM cytoplasmic localization (NPMc+), it was used as an AML lineage clonality marker. Clonal NPM exon 12 mutations were detected in myeloid, monocytic, erythroid, and megakaryocytic cells but not in fibroblasts or endothelia that were laser-microdissected from 3 patients with NPMc+ AML. Aberrant cytoplasmic expression of mutated NPM proteins was identified with anti-NPM antibodies in 2 or more myeloid hemopoietic cell lineages in 99 (61.5%) of 161 of NPMc+ AML paraffin-embedded bone marrow biopsies; lymphoid involvement was excluded in 3 investigated cases. These findings suggest that NPMc+ AML derives from either a common myeloid or earlier progenitor. Immunohistochemical studies show that varying combinations and ratios of NPMc+ leukemic cells from distinct lineages are responsible for heterogeneity within each French-American-British (FAB) classification type and for NPMc+ AML falling into different FAB categories. These findings question the value of FAB criteria in subdividing the WHO category of "AML not otherwise characterized" and suggest that, for clinical use, NPMc+ AML be provisionally regarded as a separate AML with prognostic significance.     

Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias

Nucleophosmin (NPM) is a ubiquitously expressed nucleolar phoshoprotein which shuttles continuously between the nucleus and cytoplasm. Many findings have revealed a complex scenario of NPM functions and interactions, pointing to proliferative and growth-suppressive roles of this molecule. The gene NPM1 that encodes for nucleophosmin (NPM1) is translocated or mutated in various lymphomas and leukemias, forming fusion proteins (NPM-ALK, NPM-RARalpha, NPM-MLF1) or NPM mutant products. Here, we review the structure and functions of NPM, as well as the biological, clinical and pathological features of human hematologic malignancies with NPM1 gene alterations. NPM-ALK indentifies a new category of T/Null lymphomas with distinctive molecular and clinico-pathological features, that is going to be included as a novel disease entity (ALK+ anaplastic large cell lymphoma) in the new WHO classification of lymphoid neoplasms. NPM1 mutations occur specifically in about 30% of adult de novo AML and cause aberrant cytoplasmic expression of NPM (hence the term NPMc+ AML). NPMc+ AML associates with normal karyotpe, and shows wide morphological spectrum, multilineage involvement, a unique gene expression signature, a high frequency of FLT3-internal tandem duplications, and distinctive clinical and prognostic features. The availability of specific antibodies and molecular techniques for the detection of NPM1 gene alterations has an enormous impact in the biological study diagnosis, prognostic stratification, and monitoring of minimal residual disease of various lymphomas and leukemias. The discovery of NPM1 gene alterations also represents the rationale basis for development of molecular targeted drugs.     

The applicability of the WHO classification in paediatric AML. A NOPHO‐AML study

The World Health Organization (WHO) classification of myeloid leukaemia was revised in 2008. It incorporates newly recognized entities and emphasizes the pivotal role of cytogenetic abnormalities. The aim of this study was to evaluate the usability of the WHO classification when applied to a large population‐based paediatric acute myeloid leukaemia (AML) cohort. We included children diagnosed with de novo AML, 0–18 years of age from the Nordic countries and Hong Kong from 1993 to 2012. Data were retrieved from the Nordic Society for Paediatric Haematology and Oncology AML database and patients classified according to the WHO 2008 classification. A successful karyotype was available in 97% of the cases. AML with recurrent genetic abnormalities were present in 262 (41%) and 94 (15%) were classified as AML with myelodysplasia‐related changes (AML‐MDS). WHO classifies patients with monosomy 7 and del(7q) into one group. We found that ?7 (n = 14) had significantly poorer outcome than del(7q) (n = 11); 5‐year event‐free survival 26% vs. 67%, (P = 0·02), and 5‐year overall survival 51% vs. 90%, (P = 0·04). The largest group was the highly heterogeneous AML not otherwise specified (NOS) (n = 280) (44%). In conclusion, the WHO classification allocated 15% to AML‐MDS, 44% to NOS and grouped together entities with clearly different outcome, therefore limiting the applicability of the current WHO classification in children with AML.     

上海地区805例成人急性白血病世界卫生组织亚型的临床分析

目的 分析上海地区24家医院急性白血病(AL)世界卫生组织(WHO)亚型的分布,并与国外资料进行比较,探讨我国AL患者WHO亚型的分布特点.方法 收集连续样本,对中美联合上海市白血病协作组805例AL患者同时采用FAB和WHO分型标准进行分型诊断.结果 本组AL中急性髓系白血病(AML)77.4%(623/805),急性淋巴细胞白血病(ALL)20.4%(164/805),AML和ALL之比为3.8:1.伴有重现性细胞遗传学异常AML占36.4%,AML伴多系病态造血占17.7%,不属于上述分类的AML占45.9%.FAB分型中M4最多,占33.9%,M3和M4比例高于国外,M1比例低于国外报道.与上海市1984-1994年AL资料相比,M4亚型比例增多,M1和M5亚型比例减少.AML染色体异常率61.7%,AML伴t(15;17)比例高于国外,染色体预后良好组占30.1%,预后中等组占51.6%,预后不良组占18.3%,预后良好组比例高于国外.ALL中前体B细胞ALL占绝大多数(82.3%),ALL中t(9;22)占28.2%.结论 AML伴t(15;17)和M4比例高于国外,染色体预后良好组比例高于国外,AML的WHO分型和染色体异常类型与国外有所不同.与上海市1984-1994年AL资料相比,AML的亚型分布有所变化.     

Can t(8;21) oligoblastic leukemia be called a myelodysplastic syndrome?

The new World Health Organization (WHO) classification of hematologic malignancies has incorporated t(8;21) myelodysplastic syndromes (MDS) according to the French-American-British classification into the category of acute myeloid leukemia (AML) with t(8;21)(q22;q22), while our knowledge about clinicopathological features of t(8;21) oligoblastic leukemia is still limited. We present our experience with 12 patients meeting the FAB diagnostic criteria of MDS and having t(8;21), who were compared to 43 t(8;21) AML patients. The MDS and AML patients shared most hematomorphologic, immunophenotypic, and clinical features, whereas the differences lay along myeloid maturation. The MDS patients had higher percentages of circulating neutrophils and marrow myeloid cells beyond promyelocytes than the AML patients. The incidence of Auer rods in mature neutrophils in MDS was significantly higher than that in AML, and furthermore, the neutrophils in MDS more commonly contain t(8;21) than in AML. Our findings support the rationale for the WHO classification, and future studies on large patient populations should help clarify whether the spontaneous differentiation potential could be actively associated with a hematological manifestation of t(8;21) leukemias.     

How I investigate acute myeloid leukemia

Acute myeloid leukemia (AML) is a neoplasm of immature myeloid cells and is associated with a wide variety of clinical presentations, morphological features, immunophenotypes, and genetic findings. Recent advances in identification of cytogenetic abnormalities and mutations have provided novel insights into the pathogenesis of AML. Based on the above‐mentioned parameters, the World Health Organization (WHO) classified AML into 25 subtypes, including 2 provisional entities, which differ in prognosis and treatment. In addition, certain mutations are associated with germline predisposition and increase the risk of inherited AML, which warrants family screening. Therefore, precise diagnosis and classification of AML are the most important steps in patient management. Both these steps require incorporation of history, clinical presentation, and laboratory results with studies performed by a pathologist. Pathologist‐initiated studies include morphologic evaluation on the bone marrow aspirate and/or core biopsy, immunophenotyping by flow cytometry and/or immunohistochemistry, cytogenetic analysis by karyotyping and/or fluorescence in situ hybridization, and molecular testing using gene panels and/or next‐generation sequencing. A similar approach is employed during follow‐up of patients after beginning treatment. Here, we describe in detail the various aspects of the workup, including purpose, limitations, and practice guidelines for the different studies. The process of choosing appropriate materials for the different studies is also addressed. We also provide an algorithm for the workup and risk stratification of AML based on guidelines recommended by the WHO, College of American Pathologists, National Comprehensive Cancer Network, American Society of Clinical Oncology, European Society of Medical Oncology, and the European LeukemiaNet.     

What biologic factors predict for transformation to AML?

Transformation of myelodysplastic syndromes (MDS) into secondary acute myeloid leukemia (sAML) is defined by an arbitrary boundary of ≥20% bone marrow blasts but does not necessarily reflect a defined biological transition. The more obvious distinction lies between MDS patients that have an isolated bone marrow failure phenotype and those with excess blasts. Subtyping of MDS might be more accurately stratified into clonal cytopenias and oligoblastic leukemias, using the degree of dysplasia and blast percentage as risk features, respectively, rather than as diagnostic criteria. Transformation from MDS to sAML often involves clonal evolution or expansion of existing subclones that can be assessed by changes in variant allele frequencies of the somatic mutations that define them. There are a number of predictors for transformation that have been identified: these include mutations of genes in growth signaling pathways (NRAS, KRAS, PTPN11, FLT3), mutations in genes more commonly observed in AML (NPM1, WT1, IDH2), certain cytogenetic abnormalities (monosomy 7, complex karyotype, loss of 17p). Gene expression profiles that divide MDS into two major categories identify a progenitor gene signature subtype associated with a high risk of AML transformation. Assessing for these genetic abnormalities may better identify MDS patients at greatest risk of transformation.     

A case of a primary myelofibrosis with progression and related literature review of progression phase genetics

Philadelphia (BCR-ABL)-negative myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). MPN can transform into an accelerated or a blast phase, which is associated with poor response to standard therapy and low overall median survival. We present an interesting case of a patient with a history of PMF and progression and summarize the current studies on genetic features of myeloproliferative neoplasms in blast phase (MPN-BP) with an emphasis on PMF. Although MPN-BP show ≥20% blasts in peripheral blood or bone marrow, it is not considered as acute myeloid leukemia (AML) according to the WHO classification. While MPNs-BP typically lack genetic mutations seen in de novo AML, they commonly harbor IDH1/2, SRSF2, ASXL1, and TP53 mutations, similar to the genetic profiles of acute myeloid leukemia with myelodysplasia-related changes (AML-MRC).