Single-cell mutation analysis of clonal evolution in myeloid malignancies.

Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later1-3. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.

Increased TIM-3 and galectin-9 serum levels in patients with advanced systemic mastocytosis.

BACKGROUND: Systemic mastocytosis is characterized by expansion of clonal mast cells in various tissues. Several biomarkers with diagnostic and therapeutic potential have recently been characterized in mastocytosis, such as the serum marker tryptase and the immune checkpoint molecule PD-L1. OBJECTIVE: We aimed to investigate whether serum levels of other checkpoint molecules are altered in systemic mastocytosis and whether these proteins are expressed in mastocytosis infiltrates in the bone marrow. METHODS: Levels of different checkpoint molecules were analyzed in serum of patients with different categories of systemic mastocytosis and healthy controls and correlated to disease severity. Bone marrow biopsies from patients with systemic mastocytosis were stained to confirm expression. RESULTS: Serum levels of TIM-3 and galectin-9 were increased in systemic mastocytosis, particularly in advanced subtypes, compared with healthy controls. TIM-3 and galectin-9 levels were also found to correlate with other biomarkers of systemic mastocytosis, such as serum tryptase and KIT D816V variant allele frequency in the peripheral blood. Moreover, we observed expression of TIM-3 and galectin-9 in mastocytosis infiltrates in bone marrow. CONCLUSIONS: Together, our results demonstrate for the first time that serum levels of TIM-3 and galectin-9 are increased in advanced systemic mastocytosis. Moreover, TIM-3 and galectin-9 are expressed in bone marrow infiltrates in mastocytosis. These findings provide a rationale for exploring TIM-3 and galectin-9 as diagnostic markers and eventually therapeutic targets in systemic mastocytosis, particularly in advanced forms.

[Delegation of medical duties to qualified rheumatology assistants : Effect on depression and anxiety in patients with rheumatoid arthritis]. / Delegation ärztlicher Leistungen an rheumatologische Fachassistenten : Effekte auf Depression und Angst bei Patienten mit rheumatoider Arthritis.

BACKGROUND: At least 1 comorbidity occurs in 80% of patients with rheumatoid arthritis (RA). In addition to cardiovascular comorbidities psychological comorbid conditions are common. The prevalence of depression and anxiety is higher in patients than in the general population. Screening for comorbidities is crucial. A shortage of outpatient specialist care barely allows resources for this. The implementation of team-based care holds the potential to improve the standard of care while simultaneously working against the shortage of care. OBJECTIVE: The aim of the study was to examine the effects of care on the course of depression and anxiety in patients with seropositive RA and active disease. MATERIAL AND METHODS: A multicenter pragmatic randomized controlled trial was conducted over the course of 1 year with 224 patients. After baseline, five more visits followed. In the intervention group (IG), three were initially carried out by qualified rheumatological assistants. Depression, anxiety and patient satisfaction with outpatient care were looked at in detail. RESULTS: In the IG the anxiety symptoms significantly improved over 12 months (p = 0.036). The proportions of patients with anxiety also significantly changed in the IG (p < 0.001), while there was no change in the control group between baseline and month 12. The values of the depression scale did not differ significantly (p = 0.866). In terms of the information dimension of the satisfaction questionnaire, patients in the IG felt significantly better informed after 6 months (p = 0.013) and 12 months (p = 0.003). CONCLUSION: A positive effect of team-based care on the course of depression and anxiety in patients with seropositive RA and active disease could be shown.

Which factors contribute to sexual well-being? A comparative study among 17 to 20 year old boys and girls in Belgium and Ecuador.

Despite recognition that sexual well-being is an important part of adolescent sexual and reproductive health, a clear description of adolescent sexual well-being does not yet exist. Through six in-depth interviews and four focus group discussions with 56 young people in two distinct contexts (Belgium and Ecuador), we used the social-ecological framework to identify factors influencing adolescent sexual well-being. According to respondents, the main factors that influence adolescent sexual well-being are not only situated at the individual (having knowledge and skills and being physically, sexually and mental mature and healthy) and interpersonal levels (positive attraction towards others and communication about sexuality), but at a broader societal level, including social acceptance of sex, gender and sexual diversity and its (legal) translation into comprehensive sexuality education and the ready availability of contraceptives. Our results go well beyond two existing definitions of (adolescent) sexual well-being to contribute to understanding and measurement from the perspective of young people themselves, adding substantively to ongoing discussion about the definition of the concept.

Genetic heterogeneity and clonal evolution in acute myeloid leukemia.

PURPOSE OF REVIEW: Clonal heterogeneity is a significant obstacle to successful treatment of patients with acute myeloid leukemia (AML). Here, we review new advances in the understanding of genetic heterogeneity in AML using single-cell DNA-sequencing technology. RECENT FINDINGS: New genomics and immunologic discovery tools have provided single-cell resolution maps of the clonal architecture of AML. The use of these technologies reveals the mutational landscape of AML at diagnosis, during treatment, and at relapse has an enormous degree of clonal complexity and diversity that is poised to adapt and evolve under environmental pressures. SUMMARY: AML is a complex ecosystem of competing and cooperating clones undergoing constant evolution and selection.

[Myeloproliferative neoplasms - Update on diagnosis and treatment]. / Myeloproliferative Neoplasien: Update zu Diagnostik und Therapie.

Myeloproliferative neoplasms - Update on diagnosis and treatment Abstract. Myeloproliferative neoplasms are hematopoietic stem cell disorders presenting as chronic leukemias with excessive production of mature, myeloid blood cells. Driver mutations in JAK2, CALR or MPL mediate constitutive activation of JAK2 signaling, a common hallmark of the classical Philadelphia chromosome-negative MPN including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Dysregulated hematopoiesis primarily presents with polyglobulia in PV, thrombocytosis in ET and progressive bone marrow fibrosis and increased, atypical megakaryocytes in PMF. The molecular characterization of MPNs has advanced our understanding of their pathogenesis and has facilitated diagnosis. Implementation of genetic markers enables improved prognostication, particularly in myelofibrosis. In recent years, we have seen an encouraging increase in therapeutic options for MPN including the approval of a first JAK inhibitor now followed by other agents of this class, as well as refined forms of interferon alpha. Combination therapies as well as novel therapeutic approaches are increasingly studied and hold promise for the future. Hematopoietic stem cell transplantation, which is still the only treatment option with a curative potential, is increasingly available also for elderly patients with MPN.

JAK2 exon 12 mutant mice display isolated erythrocytosis and changes in iron metabolism favoring increased erythropoiesis.

Mutations in JAK2 exon 12 are frequently found in patients with polycythemia vera (PV) that do not carry a JAK2-V617F mutation. The majority of these patients display isolated erythrocytosis. We generated a mouse model that expresses JAK2-N542-E543del, the most frequent JAK2 exon 12 mutation found in PV patients. Mice expressing the human JAK2-N542-E543del (Ex12) showed a strong increase in red blood cell parameters but normal neutrophil and platelet counts, and reduced overall survival. Erythropoiesis was increased in the bone marrow and spleen, with normal megakaryopoiesis and absence of myelofibrosis in histopathology. Erythroid progenitors and precursors were increased in hematopoietic tissues, but the numbers of megakaryocytic precursors were unchanged. Phosphorylation Stat3 and Erk1/2 proteins were increased, and a trend toward increased phospho-Stat5 and phospho-Stat1 was noted. However, Stat1 knock out in Ex12 mice induced no changes in platelet or red cell parameters, indicating that Stat1 does not play a central role in mediating the effects of Ex12 signaling on megakaryopoiesis or erythropoiesis. Ex12 mice showed decreased expression of hepcidin and increased expression of transferrin receptor-1 and erythroferrone, suggesting that the strong erythroid phenotype in Ex12 mutant mice is favored by changes in iron metabolism that optimize iron availability to allow maximal production of red cells.

'Yeah, I've grown; I can't go out anymore': differences in perceived risks between girls and boys entering adolescence.

This analysis is based on data from the Global Early Adolescent Study, which aims to understand the factors that predispose young people aged 10-14 years to positive or negative health trajectories. Specifically, interview transcripts from 202 adolescents and 191 parents across six diverse urban sites (Baltimore, Ghent, Nairobi, Ile Ife, Assuit and Shanghai) were analysed to compare the perceived risks associated with entering adolescence and how these risks differed by gender. Findings reveal that in all sites except Ghent, both young people and their parents perceived that girls face greater risks related to their sexual and reproductive health, and because of their sexual development, were perceived to require more protection. In contrast, when boys grow up, they and their parents recognised that their independence broadened, and parents felt that boys were strong enough to protect themselves. This has negative consequences as well, as boys were perceived to be more prone to risks associated with street violence and peer pressure. These differences in perceptions of vulnerability and related mobility are markers of a gender system that separates young women and men's roles, responsibilities and behaviours in ways that widen gender power imbalance with lifelong social and health consequences for people of both sexes.

A 2-way miRror of red blood cells and leukemia.

In this issue of Blood, the articles by Shaham et al and Wang et al are the first to identify microRNA 486 (miR-486) as a requisite oncomiR and credible therapeutic target in myeloid leukemia of Down syndrome (ML-DS) and chronic myeloid leukemia (CML) by showing that these 2 leukemias co-opt miR-486 functions in normal erythroid progenitor progrowth and survival activity.

Amotosalen/ultraviolet A pathogen inactivation technology reduces platelet activatability, induces apoptosis and accelerates clearance.

Amotosalen and ultraviolet A (UVA) photochemical-based pathogen reduction using the Intercept™ Blood System (IBS) is an effective and established technology for platelet and plasma components, which is adopted in more than 40 countries worldwide. Several reports point towards a reduced platelet function after Amotosalen/UVA exposure. The study herein was undertaken to identify the mechanisms responsible for the early impairment of platelet function by the IBS. Twenty-five platelet apheresis units were collected from healthy volunteers following standard procedures and split into 2 components, 1 untreated and the other treated with Amotosalen/UVA. Platelet impedance aggregation in response to collagen and thrombin was reduced by 80% and 60%, respectively, in IBS-treated units at day 1 of storage. Glycoprotein Ib (GpIb) levels were significantly lower in IBS samples and soluble glycocalicin correspondingly augmented; furthermore, GpIbα was significantly more desialylated as shown by Erythrina Cristagalli Lectin (ECL) binding. The pro-apoptotic Bak protein was significantly increased, as well as the MAPK p38 phosphorylation and caspase-3 cleavage. Stored IBS-treated platelets injected into immune-deficient nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice showed a faster clearance. We conclude that the IBS induces platelet p38 activation, GpIb shedding and platelet apoptosis through a caspase-dependent mechanism, thus reducing platelet function and survival. These mechanisms are of relevance in transfusion medicine, where the IBS increases patient safety at the expense of platelet function and survival.

Recombineering-based dissection of flanking and paralogous Hox gene functions in mouse reproductive tracts.

Hox genes are key regulators of development. In mammals, the study of these genes is greatly confounded by their large number, overlapping functions and interspersed shared enhancers. Here, we describe the use of a novel recombineering strategy to introduce simultaneous frameshift mutations into the flanking Hoxa9, Hoxa10 and Hoxa11 genes, as well as their paralogs on the HoxD cluster. The resulting Hoxa9,10,11 mutant mice displayed dramatic synergistic homeotic transformations of the reproductive tracts, with the uterus anteriorized towards oviduct and the vas deferens anteriorized towards epididymis. The Hoxa9,10,11 mutant mice also provided a genetic setting that allowed the discovery of Hoxd9,10,11 redundant reproductive tract patterning function. Both shared and distinct Hox functions were defined. Hoxd9,10,11 play a crucial role in the regulation of uterine immune function. Non-coding non-polyadenylated RNAs were among the key Hox targets, with dramatic downregulation in mutants. We observed Hox cross-regulation of transcription and splicing. In addition, we observed a surprising anti-dogmatic apparent posteriorization of the uterine epithelium. In caudal regions of the uterus, the normal simple columnar epithelium flanking the lumen was replaced by a pseudostratified transitional epithelium, normally found near the more posterior cervix. These results identify novel molecular functions of Hox genes in the development of the male and female reproductive tracts.

Genetic studies reveal an unexpected negative regulatory role for Jak2 in thrombopoiesis.

JAK inhibitor treatment is limited by the variable development of anemia and thrombocytopenia thought to be due to on-target JAK2 inhibition. We evaluated the impact of Jak2 deletion in platelets (PLTs) and megakaryocytes (MKs) on blood counts, stem/progenitor cells, and Jak-Stat signaling. Pf4-Cre-mediated Jak2 deletion in PLTs and MKs did not compromise PLT formation but caused thrombocytosis, and resulted in expansion of MK progenitors and Lin(-)Sca1(+)Kit+ cells. Serum thrombopoietin (TPO) was maintained at normal levels in Pf4-Cre-positive Jak2(f/f) mice, consistent with reduced internalization/turnover by Jak2-deficient PLTs. These data demonstrate that Jak2 in terminal megakaryopoiesis is not required for PLT production, and that Jak2 loss in PLTs and MKs results in non-autonomous expansion of stem/progenitors and of MKs and PLTs via dysregulated TPO turnover. This suggests that the thrombocytopenia frequently seen with JAK inhibitor treatment is not due to JAK2 inhibition in PLTs and MKs, but rather due to JAK2 inhibition in stem/progenitor cells.

Practical lessons for bringing policy-makers on board in sexual and reproductive health research.

BACKGROUND: The need to translate research into policy, i.e. making research findings a driving force in agenda-setting and policy change, is increasingly acknowledged. However, little is known about translation mechanisms in the field of sexual and reproductive health (SRH) outside North American or European contexts. This paper seeks to give an overview of the existing knowledge on this topic as well as to document practical challenges and remedies from the perspectives of researchers involved in four SRH research consortium projects in Latin America, sub-Saharan Africa, China and India. METHODS: A literature review and relevant project documents were used to develop an interview guide through which researchers could reflect on their experiences in engaging with policy-makers, and particularly on the obstacles met and the strategies deployed by the four project consortia to circumvent them. RESULTS: Our findings confirm current recommendations on an early and steady involvement of policy-makers, however they also suggest that local barriers between researchers and policy-making spheres and individuals can represent major hindrances to the realization of translation objectives. Although many of the challenges might be common to different contexts, creating locally-adapted responses is deemed key to overcome them. Researchers' experiences also indicate that - although inevitable - recognizing and addressing these challenges is a difficult, time- and energy-consuming process for all partners involved. Despite a lack of existing knowledge on translation efforts in SRH research outside North American or European contexts, and more particularly in low and middle-income countries, it is clear that existing pressure on health and policy systems in these settings further complicates them. CONCLUSIONS: This article brings together literature findings and researchers' own experiences in translating research results into policy and highlights the major challenges research conducted on sexual and reproductive health outside North American or European contexts can meet. Future SRH projects should be particularly attentive to these potential obstacles in order to tailor appropriate and consistent strategies within their existing resources.

Reorienting adolescent sexual and reproductive health research: reflections from an international conference.

On December 4th 2014, the International Centre for Reproductive Health (ICRH) at Ghent University organized an international conference on adolescent sexual and reproductive health (ASRH) and well-being. This viewpoint highlights two key messages of the conference--(1) ASRH promotion is broadening on different levels and (2) this broadening has important implications for research and interventions--that can guide this research field into the next decade. Adolescent sexuality has long been equated with risk and danger. However, throughout the presentations, it became clear that ASRH and related promotion efforts are broadening on different levels: from risk to well-being, from targeted and individual to comprehensive and structural, from knowledge transfer to innovative tools. However, indicators to measure adolescent sexuality that should accompany this broadening trend, are lacking. While public health related indicators (HIV/STIs, pregnancies) and their behavioral proxies (e.g., condom use, number of partners) are well developed and documented, there is a lack of consensus on indicators for the broader construct of adolescent sexuality, including sexual well-being and aspects of positive sexuality. Furthermore, the debate during the conference clearly indicated that experimental designs may not be the only appropriate study design to measure effectiveness of comprehensive, context-specific and long-term ASRH programmes, and that alternatives need to be identified and applied. Presenters at the conference clearly expressed the need to develop validated tools to measure different sub-constructs of adolescent sexuality and environmental factors. There was a plea to combine (quasi-)experimental effectiveness studies with evaluations of the development and implementation of ASRH promotion initiatives.

Sexual and gender-based violence in the European asylum and reception sector: a perpetuum mobile?

BACKGROUND: Refugees, asylum seekers and undocumented migrants are at risk of sexual and gender-based violence (SGBV) and subsequent ill-health in Europe; yet, European minimum reception standards do not address SGBV. Hence, this paper explores the nature of SGBV occurring in this sector and discusses determinants for 'Desirable Prevention'. METHODS: Applying community-based participatory research, we conducted an SGBV knowledge, attitude and practice survey with residents and professionals in eight European countries. We conducted logistic regression using mixed models to analyse the data in R. RESULTS: Of the 562 respondents, 58.3% reported cases of direct (23.3%) or peer (76.6%) victimization. Our results indicate that when men were involved, it most likely concerned sexual perpetration (adjusted odds ratio [aOR]: 4.09, confidence interval [CI]: 1.2; 13.89) and physical victimization (aOR: 2.57, CI: 1.65; 4), compared with females, who then rather perpetrated emotional violence (aOR: 1.85, CI: 1.08; 3.13) and underwent sexual victimization (aOR: 7.14, CI: 3.33; 16.67). Compared with others, asylum seekers appeared more likely to perpetrate physical (aOR 7.14, CI: 4; 12.5) and endure socio-economic violence (aOR: 10, CI: 1.37; 100), whereas professionals rather bore emotional (aOR: 2.01, CI: 0.98; 4.12) and perpetrated socio-economic violence (aOR: 25.91, CI: 13.41; 50.07). When group perpetration (aOR: 2.13, CI: 1.27; 3.58) or victimization (aOR: 1.84, CI: 1.1; 3.06) occurred, it most likely concerned socio-economic violence. CONCLUSION: Within the European asylum reception sector, residents and professionals of both sexes experience SGBV victimization and perpetration. Given the lack of prevention policies, our findings call for urgent Desirable Prevention programmes addressing determinants socio-ecologically.

Improving adolescent sexual and reproductive health in Latin America: reflections from an International Congress.

In February 2014, an international congress on Promoting Adolescent Sexual and Reproductive Health (ASRH) took place in Cuenca, Ecuador. Its objective was to share evidence on effective ASRH intervention projects and programs in Latin America, and to link this evidence to ASRH policy and program development. Over 800 people participated in the three-day event and sixty-six presentations were presented.This paper summarizes the key points of the Congress and of the Community Embedded Reproductive Health Care for Adolescents (CERCA) project. It aims at guiding future ASRH research and policy in Latin America. 1. Context matters. Individual behaviors are strongly influenced by the social context in which they occur, through determinants at the individual, relational, family, community and societal levels. Gender norms/attitudes and ease of communication are two key determinants. 2. Innovative action. There is limited and patchy evidence of effective approaches to reach adolescents with the health interventions they need at scale. Yet, there exist several promising and innovative examples of providing comprehensive sexuality education through conventional approaches and using new media, improving access to health services, and reaching adolescents as well as families and community members using community-based interventions were presented at the Congress. 3. Better measurement. Evaluation designs and indicators chosen to measure the effect and impact of interventions are not always sensitive to subtle and incremental changes. This can create a gap between measured effectiveness and the impact perceived by the targeted populations. Thus, one conclusion is that we need more evidence to better determine the factors impeding progress in ASRH in Latin American, to innovate and respond flexibly to changing social dynamics and cultural practices, and to better measure the impact of existing intervention strategies. Yet, this Congress offered a starting point from which to build a multi-agency and multi-country effort to generate specific evidence on ASRH with the aim of guiding policy and program decision-making. In a region that contains substantial barriers of access to ASRH education and services, and some of the highest adolescent pregnancy rates in the world, the participants agreed that there is no time to lose.

Sexual onset and contraceptive use among adolescents from poor neighbourhoods in Managua, Nicaragua.

BACKGROUND AND OBJECTIVES: The prevalence of teenage pregnancies in Nicaragua is the highest in Latin-America. This study aimed to gain insight into factors which determine the sexual behaviours concerned. METHODS: From July until August 2011, a door-to-door survey was conducted among adolescents living in randomly selected poor neighbourhoods of Managua. Logistic regression was used to analyse factors related to sexual onset and contraceptive use. RESULTS: Data from 2803 adolescents were analysed. Of the 475 and 299 sexually active boys and girls, 43% and 54%, respectively, reported contraceptive use. Sexual onset was positively related to increasing age, male sex, alcohol consumption and not living with the parents. Catholic boys and boys never feeling peer pressure to have sexual intercourse were more likely to report consistent condom use. Having a partner and feeling comfortable talking about sexuality with the partner were associated with hormonal contraception. CONCLUSIONS: Our data identified associates of adolescents' sexual behaviour related to personal characteristics (sex and alcohol use), to the interaction with significant others (parents, partners, peers) and to the environment (housing condition, religion). We interpreted those associates within the context of the rapidly changing society and the recently implemented health system reform in Nicaragua.