Pathogenic Mutations and Atypical Flow Cytometric Findings Characterize the Majority of Unclassifiable Myelodysplastic/Myeloproliferative Neoplasms.

OBJECTIVES: Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are a group of rare and heterogeneous hematopoietic disorders that frequently present a diagnostic challenge. Here we present our institutional experience with next-generation sequencing (NGS), together with morphologic, flow cytometric, and cytogenetic evaluation, in the diagnosis of MDS/MPN, with particular emphasis on MDS/MPN unclassifiable (MPN-U). METHODS: We evaluated the morphologic, flow cytometric, cytogenetic, and molecular characteristics of all MDS/MPN cases that underwent NGS at our institution between April 2016 and February 2019. RESULTS: Thirty-seven cases of MDS/MPN were identified, including 14 cases of MDS/MPN-U. Ninety-seven percent harbored mutations and immunophenotypic aberrancies (36/37), while only 38% had cytogenetic abnormalities (12/32). The MDS/MPN-U group had the highest rate of myeloblast phenotypic abnormalities and had a high mutation rate of approximately 2.7 mutated genes per case, most commonly in JAK2, SRSF2, and ASXL1. CONCLUSIONS: No single ancillary study was abnormal in every case, but all cases had at least one abnormal finding, demonstrating the usefulness of a multiparameter approach to the diagnosis of MDS/MPN. Although a few specific mutations were found exclusively in MDS/MPN-U and JAK2 mutations were most prevalent, larger studies are needed to determine whether MDS/MPN-U has a mutational "fingerprint," which may aid in diagnosis and targeted therapy.

MPN: The Molecular Drivers of Disease Initiation, Progression and Transformation and their Effect on Treatment.

Myeloproliferative neoplasms (MPNs) constitute a group of disorders identified by an overproduction of cells derived from myeloid lineage. The majority of MPNs have an identifiable driver mutation responsible for cytokine-independent proliferative signalling. The acquisition of coexisting mutations in chromatin modifiers, spliceosome complex components, DNA methylation modifiers, tumour suppressors and transcriptional regulators have been identified as major pathways for disease progression and leukemic transformation. They also confer different sensitivities to therapeutic options. This review will explore the molecular basis of MPN pathogenesis and specifically examine the impact of coexisting mutations on disease biology and therapeutic options.

Genomic characterization and prognostication applied to a Brazilian cohort of patients with myelofibrosis.

Genomic characterization of patients with myeloproliferative neoplasms (MPN) may lead to better diagnostic classification, prognostic assessment, and treatment decisions. These goals are particularly important in myelofibrosis (MF). We performed target Next Generation Sequencing for a panel of 255 genes and Chromosome Microarray Analysis (CMA) in 27 patients with MF. Patients were classified according to genomic findings and we compared the performance of a personalized prognostication system with IPSS, MIPSS70 and MIPSS70 + v2. Twenty-six patients presented mutations: 11.1% had single driver mutations in either JAK2, CALR or MPL; 85.2% had mutations in non-restricted genes (median: 2 per patient). CMA was abnormal in 91.7% of the 24 cases with available data. Copy-Number-Neutral Loss-of-Heterozygosity was the most common finding (66.7%). Del13q was the most frequent copy number variation, and we could define a 2.4 Mb minimally affected region encompassing RB1, SUCLA2 and CLLS2 loci. The largest genomic subgroup consisted of patients with mutations in genes involved with chromatin organization and splicing control (40.7%) and the personalized system showed better concordance and accuracy than the other prognostic systems. Comprehensive genomic characterization reveals the striking genetic complexity of MF and, when combined with clinical data, led, in our cohort, to better prognostication performance.

MDS/MPN-RS-T justified inclusion as a unique disease entity?

Myelodysplastic syndrome/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a disease entity characterized by anemia, bone marrow dysplasia with ring sideroblasts and persistent thrombocytosis ≥450 × 109/L with proliferation of large and morphologically atypical megakaryocytes. Although initially recognized by the World Health Organization only as a provisional entity, next generation sequencing has identified recurrent somatic mutations in SF3B1, JAK2 and other genes providing further evidence of the clonal nature of this disease and the need to recognize it as a separate entity. Despite its overlapping features with MDS with ring sideroblasts and essential thrombocythemia, MDS/MPN-RS-T is characterized by specific clinical features and distinct survival outcomes. In the current review we will describe the morphological and genomic features of MDS-RS-T and the potential diagnostic challenges and distinction from other possible conditions. We will also review how the current evidence supports its recognition as an independent disorder.

Myelodysplastic/myeloproliferative neoplasms: are morphology and immunophenotyping still relevant?

The term myelodysplastic/myeloproliferative neoplasm (MDS/MPN) refers to a group of clonal hematopoietic neoplasms with overlapping clinical, morphologic and genetic myelodysplastic and myeloproliferative features observed at the time of first presentation. Impaired hematopoiesis morphologically associated with evidence of myelodysplasia manifests clinically with cytopenia/s. Simultaneously, myeloproliferation is seen within the bone marrow and leads to cytosis in the peripheral blood. The diagnostic category of MDS/MPN encompasses a heterogeneous group of diseases which share similarities among them, but at the same time have distinct clinical and pathologic features and eventually diverse prognosis; such differences justify their separation in a classification scheme. In the era of genetic and genomic tests, their distinction from conventional myelodysplastic syndromes or myeloproliferative neoplasms still relies on close clinocopathological correlation, with evaluation of both peripheral blood and bone marrow samples being essential in this sense. A multiparametric integration of clinicopathologic data and cytogenetics and molecular genetics results is the preferred diagnostic approach.

Low prevalence of JAK2 V617F mutation in patients with thrombosis and normal blood counts: a retrospective impact study.

To determine the prevalence of the V617F Janus Kinase 2 (JAK2) mutation in patients with thrombosis without other biological signs of underlying myeloproliferative neoplasm (MPN) and identify associated risk factors for thrombosis. Over a 10-year period, data were collected from patients with thrombotic events and who had also been screened for the V617F JAK2 mutation. Patients with signs of underlying MPN, such as haematocrit levels ≥ 50% and/or platelet counts ≥ 450 × 109/L and/or splanchnic thrombosis were excluded from the study. Of 340 patients fulfilling inclusion criteria, JAK2 mutation was found in 9 (2.65%), the allele burden being at least 2% in 4 (1.1%). Upon follow-up, MPN was diagnosed in the latter 4. Univariate analysis of the whole cohort showed that age (54 ± 15 vs. 64 ± 13, p = 0.027), platelet count (317 ± 111 vs. 255 ± 75, p = 0.017), C-reactive protein level > 5 mg/L (OR 7.29, p = 0.014), and splenomegaly (OR 54.5, p = 0.0002) were significantly associated with JAK2 mutation. There was also a trend for an increased risk of cerebral venous thrombosis (OR 6.54, p = 0.064). Logistic regression confirmed a significant association between splenomegaly and JAK2 mutation (OR 43.15 [95%CI, 3.05-610.95], p = 0.0054). The V617F JAK2 mutation is rarely found in patients with thrombotic events without overt MPN. Splenomegaly, however, is a statistically and clinically relevant indicator of a potential JAK2 mutation in patients with non-splanchnic thrombotic events. Such patients should require further assessment and a close follow-up.

Somatic mutations and cell identity linked by Genotyping of Transcriptomes.

Defining the transcriptomic identity of malignant cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To address this challenge, here we developed Genotyping of Transcriptomes (GoT), a method to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. We apply GoT to profile 38,290 CD34+ cells from patients with CALR-mutated myeloproliferative neoplasms to study how somatic mutations corrupt the complex process of human haematopoiesis. High-resolution mapping of malignant versus normal haematopoietic progenitors revealed an increasing fitness advantage with myeloid differentiation of cells with mutated CALR. We identified the unfolded protein response as a predominant outcome of CALR mutations, with a considerable dependency on cell identity, as well as upregulation of the NF-κB pathway specifically in uncommitted stem cells. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in myeloproliferative neoplasms is dependent on the native cell identity.

Defining the Boundary Between Myelodysplastic Syndromes and Myeloproliferative Neoplasms.

In this article we provide a practical and comprehensive review of myeloid neoplasms with overlapping myelodysplastic (MDS) and myeloproliferative (MPN) features, with emphasis on recent updates in classification, particularly the utility of morphologic, cytogenetic, and molecular findings in better defining and classifying these disease entities. We provide the reader with a summary of the most recent developments and updates that have helped further our understanding of the genomic landscape, clinicopathologic features, and prognostic elements of myeloid neoplasms with MDS/MPN features.

Myelodysplastic Syndrome Updated.

This review highlights the main changes in the revised 2016 WHO Classification of Myeloid Neoplasms (published in 2017) that impact the diagnosis and management of patients with myelodysplastic syndrome (MDS). The revision was based on data accumulated since the 2008 WHO classification of MDS, much of which relates to new molecular genetic information about these neoplasms. The new information has led to some reorganization of the MDS disease categories, including a broadening of the subset of cases classified as MDS with ring sideroblasts, many of which have mutations in the spliceosome gene SF3B1. Other revisions have refined the definitions of some disease categories to improve disease risk stratification. The revised categories in the new classification ensure that MDS patients receive risk-adapted therapies based on the most recently available data.

The Diagnostic Work-Up of Hypereosinophilia.

Hypereosinophilia (HE) is defined as a persistent elevated eosinophil count of ≥1.5 × 109/L. HE can be one of the dominant manifestations of a hematopoietic myeloid neoplasm or secondary/reactive to an underlying medical condition. If a cause of HE and its associated tissue/organ damage is not determined, the condition is considered to be idiopathic hypereosinophilic syndrome (HES). The work-up of HE can be challenging due to a broad range of causes of HE that can be either reactive or neoplastic. In recent years, with the advent of molecular genetic testing and the introduction of targeted therapy in the management of these patients, there is a growing interest in better characterization of these diseases. Using a multimodality approach and following a proper -algorithm, a diagnosis can be made in a large proportion of patients. In idiopathic HES, myeloid neoplasm associated -somatic mutations as evidence of clonality are reported in -20-25% patients; however, the mutation data should be -interpreted cautiously considering the prevalence of clonal hematopoiesis of indeterminate potential (CHIP). Bone marrow morphology has been shown to have important value in the identification of a true myeloid neoplasm in these disorders. A genome-wide study may be needed to understand the "idiopathic" cases that would ultimately lead to better patient care.

Myeloid Neoplasms with Germline Predisposition.

The updated 2016 WHO classification of hematopoietic tumors has a new category: "myeloid neoplasms with germline predisposition." These entities are rare, but are also currently underdiagnosed and underreported. Recognition is critical for appropriate clinical evaluation and therapy, with potential implications for the patient's entire family. The WHO includes 3 categories of myeloid neoplasms with germline predisposition: neoplasms without preexisting conditions, neoplasms with a history of thrombocytopenia, and neoplasms with other organ dysfunction. Specialized molecular testing is frequently necessary to make the diagnosis, as the presence of one of the implicated mutations is not sufficient for diagnosis and should be confirmed with germline DNA evaluation. Many families have unique mutations that are not detected by targeted sequencing panels. Periodic bone marrow (BM) examinations are recommended to assess patients' baseline morphology and rule out evidence of disease progression. Thus, accurate diagnosis requires a careful recording of clinical history, a BM morphology evaluation, and advanced molecular testing.

[A revised 4 edition WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues, 2017: myeloid neoplasms]. / Peresmotrennaia Klassifikatsiia VOZ opukholei gemopoéticheskoi i limfoidnoi tkani, 2017 (4-e izdanie): mieloidnye neoplazii.

The paper presents new molecular data, the principles of the classification of myeloid neoplasms, and criteria for their diagnosis according to the new edition of the WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues, 2017. Current concepts of clonal hematopoiesis and models of clonal evolution are presented to characterize the common features of the molecular pathogenesis of myeloid neoplasms. There are new data and general principles of diagnosis of myeloid neoplasms: Ph-negative myeloproliferative diseases, myelodysplastic syndromes, myeloid/lymphoid neoplasms with eosinophilia and rearrangements of PDGFRA, PDGFRB, FGFR1, and PCM1-JAK2, diseases from the group of myelodysplastic/myeloproliferative diseases. Emphasis is laid on the possibilities and limitations of pathological differential diagnosis when a pathologist examines bone marrow trepanobiopsy specimens in his/her routine work.

Classification and Personalized Prognosis in Myeloproliferative Neoplasms.

BACKGROUND: Myeloproliferative neoplasms, such as polycythemia vera, essential thrombocythemia, and myelofibrosis, are chronic hematologic cancers with varied progression rates. The genomic characterization of patients with myeloproliferative neoplasms offers the potential for personalized diagnosis, risk stratification, and treatment. METHODS: We sequenced coding exons from 69 myeloid cancer genes in patients with myeloproliferative neoplasms, comprehensively annotating driver mutations and copy-number changes. We developed a genomic classification for myeloproliferative neoplasms and multistage prognostic models for predicting outcomes in individual patients. Classification and prognostic models were validated in an external cohort. RESULTS: A total of 2035 patients were included in the analysis. A total of 33 genes had driver mutations in at least 5 patients, with mutations in JAK2, CALR, or MPL being the sole abnormality in 45% of the patients. The numbers of driver mutations increased with age and advanced disease. Driver mutations, germline polymorphisms, and demographic variables independently predicted whether patients received a diagnosis of essential thrombocythemia as compared with polycythemia vera or a diagnosis of chronic-phase disease as compared with myelofibrosis. We defined eight genomic subgroups that showed distinct clinical phenotypes, including blood counts, risk of leukemic transformation, and event-free survival. Integrating 63 clinical and genomic variables, we created prognostic models capable of generating personally tailored predictions of clinical outcomes in patients with chronic-phase myeloproliferative neoplasms and myelofibrosis. The predicted and observed outcomes correlated well in internal cross-validation of a training cohort and in an independent external cohort. Even within individual categories of existing prognostic schemas, our models substantially improved predictive accuracy. CONCLUSIONS: Comprehensive genomic characterization identified distinct genetic subgroups and provided a classification of myeloproliferative neoplasms on the basis of causal biologic mechanisms. Integration of genomic data with clinical variables enabled the personalized predictions of patients' outcomes and may support the treatment of patients with myeloproliferative neoplasms. (Funded by the Wellcome Trust and others.).

What is pre-fibrotic myelofibrosis and how should it be managed in 2018?

The 2016 World Health Organization (WHO) classification for myeloproliferative neoplasms (MPN) divided myelofibrosis (MF) into pre-fibrotic (pre-MF) and overt-MF categories. This new classification, particularly the entity pre-MF, has been a subject of discussion between experts. Important questions have been raised in recent years, such as the need for bone marrow trephine for diagnosis; how this is interpreted and the weighting given to it in assigning a diagnosis; determination of prognosis for pre-MF patients; including which scoring system to use and, ultimately, an evidence-based management plan for this group of patients. Many pre-MF patients present as young adults, with thrombocytosis, elevated lactate dehydrogenase levels and increased bone marrow fibrosis (i.e. ≥ grade 1). Current management strategies differ in view of age, comorbidities and bone marrow features and the opinion of the managing clinicians. Prognostic scoring systems have some limitations regarding this entity, and at the present time there is limited information about the overall survival and incidence of progression to overt-MF and acute leukaemia for pre-MF. In this clinically focussed article, we review the main characteristics of this new disease category in view of the current published literature and illustrate our discussion with some real patient cases. Lastly, we propose a management strategy for patients to whom this diagnostic label is applied.

[Myeloproliferative neoplasms: revisions in the 2016 WHO criteria].

The 2016 revised WHO classification of tumors of hematopoietic and lymphoid tissues has incorporated novel molecular markers, such as calreticulin (CALR) mutations, for the diagnosis of myeloproliferative neoplasms (MPNs). Typically, CALR mutations are detected in 25%-30% of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) and can lead to frameshifts that produce proteins with a novel C-terminal. In addition, the CALR mutation plays a crucial role in the MPN pathogenesis. The second major revision comprises the change in RBC parameters for polycythemia diagnosis; additionally, it emphasizes BM biopsy for the diagnosis of PV. Previously, PV was often underdiagnosed when considering the Hb levels of >18.5 g/dl for males and >16.5 g/dl for females; thus, the 2016 revision lowered these levels to >16.5 g/dl for males and >16.0 g/l for females. The third major revision is the introduction of a novel entity "prefibrotic/early" PMF (prePMF) to PMF. Although megakaryocytic proliferation and atypia were observed in in BM biopsy specimens of prePMF, these were not accompanied by reticulin fibrosis > grade 1. Thus, the inferior prognosis of prePMF was reported in comparison with "true" ET.

Early/prefibrotic primary myelofibrosis in patients who were initially diagnosed with essential thrombocythemia.

A new entity, namely early/prefibrotic primary myelofibrosis (PMF), was introduced as a subtype of PMF in the 2016 revised World Health Organization (WHO) criteria for myeloproliferative neoplasms (MPN). It was diagnosed based on histopathological features of bone marrow (BM) biopsy specimens together with clinical parameters [leukocytosis, anemia, elevated lactate dehydrogenase (LDH) values, and splenomegaly]. The aim of this study was to evaluate the prevalence of early/prefibrotic PMF in patients who were previously diagnosed with ET, and to compare clinical features at diagnosis and outcomes between early/prefibrotic PMF and essential thrombocythemia (ET) patients. BM biopsy samples obtained at the time of ET diagnosis were available in 42 patients. Sample reevaluation according to the 2016 revised WHO criteria revealed that early/prefibrotic PMF accounted for 14% of patients who were previously diagnosed with ET, which was comparable to the rates in previous reports. Compared to patients with ET, patients with early/prefibrotic PMF had higher LDH values and higher frequencies of splenomegaly. Overall, myelofibrosis-free and acute myeloid leukemia-free survivals were comparable between the 2 groups. Accurate diagnosis is required to clarify the clinical features of Japanese ET patients.

Recent advances in the genomics and therapy of BCR/ABL1-positive and -negative chronic myeloproliferative neoplasms.

This review is based on the presentations and deliberations at the 7th John Goldman Chronic Myeloid Leukemia (CML) and Myeloproliferative Neoplasms (MPN) Colloquium which took place in Estoril, Portugal on the 15th October 2017, and the 11th post-ASH International Workshop on CML and MPN which took place on the 6th-7th December 2016, immediately after the 58th American Society of Hematology Annual Meeting. Rather than present a resume of the proceedings, we have elected to address some of the topical translational research and clinically relevant topics in greater detail. We address recent updates in the genetics and epigenetics of MPN, the mechanisms of transformation by mutant calreticulin, advances in the biology and therapy of systemic mastocytosis, clinical updates on JAK2 inhibitors and other therapeutic approaches for patients with MPNs, cardiovascular toxicity related to tyrosine kinase inhibitors and the concept of treatment-free remission for patients with CML.