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The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE−/− mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal disorders involving hematopoietic stem and progenitor cells and are associated with myeloproliferation, splenomegaly and constitutional symptoms. Similar signs and symptoms can also be found in patients with chronic inflammatory diseases, and inflammatory processes have been found to play an important role in the pathogenesis and progression of MPNs. Signal transduction pathways involving JAK1, JAK2, STAT3 and STAT5 are causally involved in driving both the malignant cells and the inflammatory process. Moreover, anti-inflammatory and immune-modulating drugs have been used successfully in the treatment of MPNs. However, to date, many unresoved issues remain. These include the role of somatic mutations that are present in addition to JAK2V617F, CALR and MPL W515 mutations, the interdependency of malignant and nonmalignant cells and the means to eradicate MPN-initiating and -maintaining cells. It is imperative for successful therapeutic approaches to define whether the malignant clone or the inflammatory cells or both should be targeted. The present review will cover three aspects of the role of inflammation in MPNs: inflammatory states as important differential diagnoses in cases of suspected MPN (that is, in the absence of a clonal marker), the role of inflammation in MPN pathogenesis and progression and the use of anti-inflammatory drugs for MPNs. The findings emphasize the need to separate the inflammatory processes from the malignancy in order to improve our understanding of the pathogenesis, diagnosis and treatment of patients with Philadelphia-negative MPNs.

Although acquired mutations in KIT are commonly detected in various categories of mastocytosis, the methodologies applied to detect and quantify the mutant type and allele burden in various cells and tissues are poorly defined. We here propose a consensus on methodologies used to detect KIT mutations in patients with mastocytosis at diagnosis and during follow-up with sufficient precision and sensitivity in daily practice. In addition, we provide recommendations for sampling and storage of diagnostic material as well as a robust diagnostic algorithm. Using highly sensitive assays, KIT D816V can be detected in peripheral blood leukocytes from most patients with systemic mastocytosis (SM) that is a major step forward in screening and SM diagnosis. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy. Our recommendations should greatly facilitate diagnostic and follow-up investigations in SM in daily practice as well as in clinical trials. In addition, the new tools and algorithms proposed should lead to a more effective screen, early diagnosis of SM and help to avoid unnecessary referrals.

Most patients with KIT D816V + advanced systemic mastocytosis (SM) are characterized by somatic mutations in additional genes. We sought to clarify the prognostic impact of such mutations. Genotype and clinical characteristics of 70 multi-mutated KIT D816V + advanced SM patients were included in univariate and multivariate analyses. The most frequently identified mutated genes were TET2 ( n =33 of 70 patients), SRSF2 ( n =30), ASXL1 ( n =20), RUNX1 ( n =16) and JAK2 ( n =11). In univariate analysis, overall survival (OS) was adversely influenced by mutations in SRSF2 ( P <0.0001), ASXL1 ( P =0.002) and RUNX1 ( P =0.03), but was not influenced by mutations in TET2 or JAK2 . In multivariate analysis, SRSF2 and ASXL1 remained the most predictive adverse indicators concerning OS. Furthermore, we found that inferior OS and adverse clinical characteristics were significantly influenced by the number of mutated genes in the SRSF2 / ASXL1 / RUNX1 (S/A/R) panel ( P <0.0001). In conclusion, the presence and number of mutated genes within the S/A/R panel are adversely associated with advanced disease and poor survival in KIT D816V + SM. On the basis of these findings, inclusion of molecular markers should be considered in upcoming prognostic scoring systems for patients with SM.

The signal transducer and activator of transcription 5 (STAT5) regulates differentiation, survival, proliferation and transformation of hematopoietic cells. Upon cytokine stimulation, STAT5 tyrosine phosphorylation (pYSTAT5) is transient, while in diverse neoplastic cells persistent overexpression and enhanced pYSTAT5 are frequently found. Post-translational modifications might contribute to enhanced STAT5 activation in the context of transformation, but the strength and duration of pYSTAT5 are incompletely understood. We found that O-GlcNAcylation and tyrosine phosphorylation act together to trigger pYSTAT5 levels and oncogenic transcription in neoplastic cells. The expression of a mutated hyperactive gain-of-function (GOF) STAT5 without O-GlcNAcylation resulted in decreased tyrosine phosphorylation, oligomerization and transactivation potential and complete loss of oncogenic transformation capacity. The lack of O-GlcNAcylation diminished phospho-ERK and phospho-AKT levels. Our data show that O-GlcNAcylation of STAT5 is an important process that contributes to oncogenic transcription through enhanced STAT5 tyrosine phosphorylation and oligomerization driving myeloid transformation. O-GlcNAcylation of STAT5 could be required for nutrient sensing and metabolism of cancer cells. Leukemia (2017) 31, 2132-2142; doi: 10.1038/leu.2017.4; published online 10 February 2017

Basophils form a distinct cell lineage within the hematopoietic cell family. In various myeloid neoplasms, including chronic myeloid leukemia, basophilia is frequently seen. Acute and chronic basophilic leukemias, albeit rare, have also been described. However, no generally accepted criteria and classification of basophilic leukemias have been presented to date. To address this unmet need, a series of Working Conferences and other meetings were organized between March 2015 and March 2016. The current article provides a summary of consensus statements from these meetings, together with proposed criteria to delineate acute basophilic leukemia (ABL) from chronic basophilic leukemia (CBL) and primary forms of the disease where no preceding myeloid malignancy is detected, from the more common ‘secondary’ variants. Moreover, the term hyperbasophilia (HB) is proposed for cases with a persistent peripheral basophil count ⩾1000 per μl of blood. This condition, HB, is highly indicative of the presence of an underlying myeloid neoplasm. Therefore, HB is an important checkpoint in the diagnostic algorithm and requires a detailed hematologic investigation. In these patients, an underlying myeloid malignancy is often found and is then labeled with the appendix -baso, whereas primary cases of ABL or CBL are very rare. The criteria and classification proposed in this article should facilitate the diagnosis and management of patients with unexplained basophilia and basophil neoplasms in routine practice, and in clinical studies.

We evaluated the impact of clinical and molecular characteristics on overall survival (OS) in 108 patients with indolent ( n =41) and advanced systemic mastocytosis (SM) (advSM, n =67). Organomegaly was measured by magnetic resonance imaging-based volumetry of the liver and spleen. In multivariate analysis of all patients, an increased spleen volume ⩾450 ml (hazard ratio (HR), 5.2; 95% confidence interval (CI), (2.1–13.0); P =0.003) and an elevated alkaline phosphatase (AP; HR 5.0 (1.1–22.2); P =0.02) were associated with adverse OS. The 3-year OS was 100, 77, and 39%, respectively ( P <0.0001), for patients with 0 (low risk, n =37), 1 (intermediate risk, n =32) or 2 (high risk, n =39) parameters. For advSM patients with fully available clinical and molecular data ( n =60), univariate analysis identified splenomegaly ⩾1200 ml, elevated AP and mutations in the SRSF2 / ASXL1 / RUNX1 (S/A/R) gene panel as significant prognostic markers. In multivariate analysis, mutations in S/A/R (HR 3.2 (1.1–9.6); P =0.01) and elevated AP (HR 2.6 (1.0–7.1); P =0.03) remained predictive adverse prognostic markers for OS. The 3-year OS was 76 and 38%, respectively ( P =0.0003), for patients with 0–1 (intermediate risk, n =28) or 2 (high risk, n =32) parameters. We conclude that splenomegaly, elevated AP and mutations in the S/A/R gene panel are independent of the World Health Organization classification and provide the most relevant prognostic information in SM patients.

To explore the molecular profile and its prognostic implication in systemic mastocytosis (SM), we analyzed the mutation status of granulocyte–macrophage colony-forming progenitor cells (CFU-GM) in patients with KIT D816V + indolent SM (ISM, n =4), smoldering SM (SSM, n =2), aggressive SM (ASM, n =1), SM with associated clonal hematologic non-mast cell lineage disorder (SM-AHNMD, n =5) and ASM-AHNMD ( n =7). All patients with (A)SM-AHNMD ( n =12) carried 1–4 (median 3) additional mutations in 11 genes tested, most frequently TET2 , SRSF2 , ASXL1 , CBL and EZH2 . In multi-mutated (A)SM-AHNMD, KIT D816V + single-cell-derived CFU-GM colonies were identified in 8/12 patients (median 60%, range 0–95). Additional mutations were identified in CFU-GM colonies in all patients, and logical hierarchy analysis indicated that mutations in TET2 , SRSF2 and ASXL1 preceded KIT D816V. In ISM/SSM, no additional mutations were detected and CFU-GM colonies were exclusively KIT D816V − . These data indicate that (a) (A)SM-AHNMD is a multi-mutated neoplasm, (b) mutations in TET2 , SRSF2 or ASXL1 precede KIT D816V in ASM-AHNMD, (c) KIT D816V is thus a phenotype modifier toward SM and (d) KIT D816V or other mutations are rare in CFU-GM colonies of ISM/SSM patients, which might explain at least in part their better prognosis.

The detailed molecular mechanism of action of second-generation BCR–ABL tyrosine kinase inhibitors, including perturbed targets and pathways, should contribute to rationalized therapy in chronic myeloid leukemia (CML) or in other affected diseases. Here, we characterized the target profile of the dual SRC/ABL inhibitor bosutinib employing a two-tiered approach using chemical proteomics to identify natural binders in whole cell lysates of primary CML and K562 cells in parallel to in vitro kinase assays against a large recombinant kinase panel. The combined strategy resulted in a global survey of bosutinib targets comprised of over 45 novel tyrosine and serine/threonine kinases. We have found clear differences in the target patterns of bosutinib in primary CML cells versus the K562 cell line. A comparison of bosutinib with dasatinib across the whole kinase panel revealed overlapping, but distinct, inhibition profiles. Common among those were the SRC, ABL and TEC family kinases. Bosutinib did not inhibit KIT or platelet-derived growth factor receptor, but prominently targeted the apoptosis-linked STE20 kinases. Although in vivo bosutinib is inactive against ABL T315I, we found this clinically important mutant to be enzymatically inhibited in the mid-nanomolar range. Finally, bosutinib is the first kinase inhibitor shown to target CAMK2G, recently implicated in myeloid leukemia cell proliferation.

A risk-adapted treatment strategy is mandatory for myelodysplastic syndromes (MDS). We refined the World Health Organization (WHO)-classification-based Prognostic Scoring System (WPSS) by determining the impact of the newer clinical and cytogenetic features, and we compared its prognostic power to that of the revised International Prognostic Scoring System (IPSS-R). A population of 5326 untreated MDS was considered. We analyzed single WPSS parameters and confirmed that the WHO classification and severe anemia provide important prognostic information in MDS. A strong correlation was found between the WPSS including the new cytogenetic risk stratification and WPSS adopting original criteria. We then compared WPSS with the IPSS-R prognostic system. A highly significant correlation was found between the WPSS and IPSS-R risk classifications. Discrepancies did occur among lower-risk patients in whom the number of dysplastic hematopoietic lineages as assessed by morphology did not reflect the severity of peripheral blood cytopenias and/or increased marrow blast count. Moreover, severe anemia has higher prognostic weight in the WPSS versus IPSS-R model. Overall, both systems well represent the prognostic risk of MDS patients defined by WHO morphologic criteria. This study provides relevant in formation for the implementation of risk-adapted strategies in MDS.