Community effort endorsing multiscale modelling, multiscale data science and multiscale computing for systems medicine.

Systems medicine holds many promises, but has so far provided only a limited number of proofs of principle. To address this road block, possible barriers and challenges of translating systems medicine into clinical practice need to be identified and addressed. The members of the European Cooperation in Science and Technology (COST) Action CA15120 Open Multiscale Systems Medicine (OpenMultiMed) wish to engage the scientific community of systems medicine and multiscale modelling, data science and computing, to provide their feedback in a structured manner. This will result in follow-up white papers and open access resources to accelerate the clinical translation of systems medicine.

Diagnosis and management of non-clonal erythrocytosis remains challenging: a single centre clinical experience.

Erythrocytosis has a diverse background. While polycythaemia vera has well defined criteria, the diagnostic approach and management of other types of erythrocytosis are more challenging. The aim of study was to retrospectively analyse the aetiology and management of non-clonal erythrocytosis patients referred to a haematology outpatient clinic in an 8-year period using a 3-step algorithm. The first step was inclusion of patients with Hb > 185 g/L and/or Hct > 0.52 in men and Hb > 165 g/L and/or Hct > 0.48 in women on two visits ≥ two months apart, thus confirming true erythrocytosis. Secondly, polycythaemia vera was excluded and secondary causes of erythrocytosis (SE) identified. Thirdly, idiopathic erythrocytosis patients (IE) were referred to next-generation sequencing for possible genetic background evaluation. Of the 116 patients, 75 (65%) are men and 41 (35%) women, with non-clonal erythrocytosis 34/116 (29%) had SE, 15/116 (13%) IE and 67/116 (58%) stayed incompletely characterized (ICE). Patients with SE were significantly older and had significantly higher Hb and Hct compared to patients with IE. Most frequently, SE was attributed to obstructive sleep apnoea and smoking. Phlebotomies were performed in 56, 53 and 40% of patients in the SE, IE, and ICE group, respectively. Approx. 70% of patients in each group received aspirin. Thrombotic events were registered in 12, 20 and 15% of SE, IE and ICE patients, respectively. Congenital erythrocytosis type 4 (ECYT4) was diagnosed in one patient. The study demonstrates real-life management of non-clonal erythrocytosis which could be optimized using a 3-step diagnostic algorithm.

Genetic analysis of 39 erythrocytosis and hereditary hemochromatosis-associated genes in the Slovenian family with idiopathic erythrocytosis.

BACKGROUND: Erythrocytosis is a condition with an excessive number of erythrocytes, accompanied by an elevated haemoglobin and/or haematocrit value. Congenital erythrocytosis has a diverse genetic background with several genes involved in erythropoiesis. In clinical practice, nine genes are usually examined, but in approximately 70% of patients, no causative mutation can be identified. In this study, we screened 39 genes, aiming to identify potential disease-driving variants in the family with erythrocytosis of unknown cause. PATIENTS AND METHODS: Two affected family members with elevated haemoglobin and/or haematocrit and negative for acquired causes and one healthy relative from the same family were selected for molecular-genetic analysis of 24 erythrocytosis and 15 hereditary haemochromatosis-associated genes with targeted NGS. The identified variants were further analysed for pathogenicity using various bioinformatic tools and review of the literature. RESULTS: Of the 12 identified variants, two heterozygous variants, the missense variant c.471G>C (NM_022051.2) (p.(Gln157His)) in the EGLN1 gene and the intron variant c.2572-13A>G (NM_004972.3) in the JAK2 gene, were classified as low-frequency variants in European population. None of the two variants were present in a healthy family member. Variant c.2572-13A>G has potential impact on splicing by one prediction tool. CONCLUSION: For the first time, we included 39 genes in the erythrocytosis clinical panel and identified two potential disease-driving variants in the Slovene family studied. Based on the reported functional in vitro studies combined with our bioinformatics analysis, we suggest further functional analysis of variant in the JAK2 gene and evaluation of a cumulative effect of both variants.

Molecular Insights into the Oxygen-Sensing Pathway and Erythropoietin Expression Regulation in Erythropoiesis.

Erythropoiesis is regulated by several factors, including the oxygen-sensing pathway as the main regulator of erythropoietin (EPO) synthesis in the kidney. The release of EPO from the kidney and its binding to the EPO receptor (EPOR) on erythrocyte progenitor cells in the bone marrow results in increased erythropoiesis. Any imbalance in these homeostatic mechanisms can lead to dysregulated erythropoiesis and hematological disorders. For example, mutations in genes encoding key players of oxygen-sensing pathway and regulation of EPO production (HIF-EPO pathway), namely VHL, EGLN, EPAS1 and EPO, are well known causative factors that contribute to the development of erythrocytosis. We aimed to investigate additional molecular mechanisms involved in the HIF-EPO pathway that correlate with erythropoiesis. To this end, we conducted an extensive literature search and used several in silico tools. We identified genes encoding transcription factors and proteins that control transcriptional activation or repression; genes encoding kinases, deacetylases, methyltransferases, conjugating enzymes, protein ligases, and proteases involved in post-translational modifications; and genes encoding nuclear transport receptors that regulate nuclear transport. All these genes may modulate the stability or activity of HIF2α and its partners in the HIF-EPO pathway, thus affecting EPO synthesis. The theoretical information we provide in this work can be a valuable tool for a better understanding of one of the most important regulatory pathways in the process of erythropoiesis. This knowledge is necessary to discover the causative factors that may contribute to the development of hematological diseases and improve current diagnostic and treatment solutions in this regard.

STAT5 as a Key Protein of Erythropoietin Signalization.

Erythropoietin (EPO) acts on multiple tissues through its receptor EPOR, a member of a cytokine class I receptor superfamily with pleiotropic effects. The interaction of EPO and EPOR triggers the activation of several signaling pathways that induce erythropoiesis, including JAK2/STAT5, PI3K/AKT, and MAPK. The canonical EPOR/JAK2/STAT5 pathway is a known regulator of differentiation, proliferation, and cell survival of erythroid progenitors. In addition, its role in the protection of other cells, including cancer cells, is under intense investigation. The involvement of EPOR/JAK2/STAT5 in other processes such as mRNA splicing, cytoskeleton reorganization, and cell metabolism has been recently described. The transcriptomics, proteomics, and epigenetic studies reviewed in this article provide a detailed understanding of EPO signalization. Advances in this area of research may be useful for improving the efficacy of EPO therapy in hematologic disorders, as well as in cancer treatment.

The Role of PI3K/AKT and MAPK Signaling Pathways in Erythropoietin Signalization.

Erythropoietin (EPO) is a glycoprotein cytokine known for its pleiotropic effects on various types of cells and tissues. EPO and its receptor EPOR trigger signaling cascades JAK2/STAT5, MAPK, and PI3K/AKT that are interconnected and irreplaceable for cell survival. In this article, we describe the role of the MAPK and PI3K/AKT signaling pathways during red blood cell formation as well as in non-hematopoietic tissues and tumor cells. Although the central framework of these pathways is similar for most of cell types, there are some stage-specific, tissue, and cell-lineage differences. We summarize the current state of research in this field, highlight the novel members of EPO-induced PI3K and MAPK signaling, and in this respect also the differences between erythroid and non-erythroid cells.

Methylation of the first exon in the erythropoietin receptor gene does not correlate with its mRNA and protein level in cancer cells.

BACKGROUND: Erythropoietin receptor (EPOR) is a functional membrane-bound cytokine receptor. Erythropoietin (EPO) represents an important hematopoietic factor for production, maturation and differentiation of erythroid progenitors. In non-hematopoietic tissue, EPO/EPOR signalization could also play cytoprotective and anti-apoptotic role. Several studies identified pro-stimulating EPO/EPOR effects in tumor cells; however, numerous studies opposed this fact due to the usage of unspecific EPOR antibodies and thus potential absence or very low levels of EPOR in tumor cells. It seems that this problem is more complex and therefore we have decided to focus on EPOR expression at several levels such as the role of methylation in the regulation of EPOR expression, identification of possible EPOR transcripts and the presence of EPOR protein in selected tumor cells. METHODS: Methylation status was analysed by bisulfite conversion reaction, PCR and sequencing. The expression of EPOR was monitored by quantitative RT-PCR and western blot analysis. RESULTS: In this study we investigated the methylation status of exon 1 of EPOR gene in selected human cancer cell lines. Our results indicated that CpGs methylation in exon 1 do not play a significant role in the regulation of EPOR transcription. However, methylation status of EPOR exon 1 was cell type dependent. We also observed the existence of two EPOR splice variants in human ovarian adenocarcinoma cell line - A2780 and confirmed the expression of EPOR protein in these cells using specific A82 anti-EPOR antibody. CONCLUSION: We outlined the methylation status of all selected cancer cell lines in exon 1 of EPOR gene and these results could benefit future investigations. Moreover, A82 antibody confirmed our previous results demonstrating the presence of functional EPOR in human ovarian adenocarcinoma A2780 cells.

Genetic variability of hypoxia-inducible factor alpha (HIFA) genes in familial erythrocytosis: Analysis of the literature and genome databases.

Familial erythrocytosis (FE) is a congenital disorder, defined by elevated red blood cell number, hemoglobin, and hematocrit. Among eight types of FE, type 4 is caused by variants in the EPAS1 gene. Two other hypoxia-inducible factor alpha (HIFA) subunits, HIF1A and HIF3A, have not yet been associated with medical history of FE, but have potential role in the development of erythrocytosis. To improve diagnosis, it is crucial to identify new variants in genes involved in erythrocyte production. Published literature and data from genome browsers were used to obtain HIFA sequence variants associated with erythrocytosis and to locate them on protein sequence and regulatory sites. We retrieved 24 variants from the literature: 2 in HIF1A, 20 in EPAS1 and 2 in HIF3A gene. Sixteen out of 20 variants in the EPAS1 gene are positioned in a conserved region of 13 amino acids within exon 12, next to regulatory post-translational modification and binding sites, suggesting that EPAS1 has an important role in erythropoiesis. The role of HIF1A and HIF3A in the development of erythrocytosis should be further investigated.

Erythropoietin and Its Angiogenic Activity.

Erythropoietin (EPO) is the main hematopoietic hormone acting on progenitor red blood cells via stimulation of cell growth, differentiation, and anti-apoptosis. However, its receptor (EPOR) is also expressed in various non-hematopoietic tissues, including endothelium. EPO is a pleiotropic growth factor that exhibits growth stimulation and cell/tissue protection on numerous cells and tissues. In this article we review the angiogenesis potential of EPO on endothelial cells in heart, brain, and leg ischemia, as well as its role in retinopathy protection and tumor promotion. Furthermore, the effect of EPO on bone marrow and adipose tissue is also discussed.

Cannabinoids and triple-negative breast cancer treatment.

Triple-negative breast cancer (TNBC) accounts for about 10-20% of all breast cancer cases and is associated with an unfavorable prognosis. Until recently, treatment options for TNBC were limited to chemotherapy. A new successful systemic treatment is immunotherapy with immune checkpoint inhibitors, but new tumor-specific biomarkers are needed to improve patient outcomes. Cannabinoids show antitumor activity in most preclinical studies in TNBC models and do not appear to have adverse effects on chemotherapy. Clinical data are needed to evaluate efficacy and safety in humans. Importantly, the endocannabinoid system is linked to the immune system and immunosuppression. Therefore, cannabinoid receptors could be a potential biomarker for immune checkpoint inhibitor therapy or a novel mechanism to reverse resistance to immunotherapy. In this article, we provide an overview of the currently available information on how cannabinoids may influence standard therapy in TNBC.

Utility of next-generation sequencing in identifying congenital erythrocytosis in patients with idiopathic erythrocytosis.

Background: Congenital erythrocytosis (CE) is increasingly recognized as the cause of erythrocytosis in patients in whom polycythemia vera and secondary acquired causes have been excluded. The aim of our study was to determine possible genetic background in patients with idiopathic erythrocytosis. Methods: 40 patients with idiopathic erythrocytosis, referred to our institution in a 5-year period, were analyzed. We collected data on erythropoietin (Epo) levels, hemoglobin (Hgb), hematocrit (Hct), erythrocyte count, age, gender, past thrombotic events, concomitant diseases, and smoking status. CE was tested using next-generation sequencing (NGS), in the majority of patients also measurement of P50 and Hgb electrophoresis were performed. Patients with signs of iron overload were tested for genetic variants in the HFE gene. Results: The median patient age at analysis was 46.5 years (range 22-73), with 37 out of 40 being males (93 %). The median Hgb, Hct and red blood cells count were 180 g/L, 0.51, 5.985 x 1012/L in men and 171 g/L, 0.50 and 5.68 x 1012/L in women, respectively. Epo levels were decreased in three, increased in one patient and within the normal range in the rest (median 7.55 mIU/mL; range 2.90-19.50). Eight patients (20 %) smoked. 32 (80 %) were treated with low-dose aspirin, and 20 (50 %) underwent at least one phlebotomy. Thromboembolic events were recorded in 2 patients (5 %). P50 was measured in 20 out of 40 patients, and it was above 24 mm Hg (3.12 kPa) in all of them. Hemoglobin electrophoresis was performed in 73 % of patients, with no abnormal Hgb detected. Variants in the HFE gene were found in 8 out of 40 patients (20 %), but in only one patient the results were associated with an increased risk for hemochromatosis. Although no pathogenic variants for CE were detected by NGS, two variants of uncertain significance, namely EGLN1 (NM_022051.2):c.1072C>T (p.(Pro358Ser)) and EGLN1 (NM_022051.2):c.1124A>G (p.(Glu375Gly)) were identified as strong etiologic candidates. Conclusion: CE is an extremely rare condition. Genetic testing is advised in young individuals with a long-standing persistent erythrocytosis, possibly with a family history and after exclusion of more frequent secondary causes and polycytemia vera.

Recombinant human erythropoietin alters gene expression and stimulates proliferation of MCF-7 breast cancer cells.

BACKGROUND: Functional erythropoietin (EPO) signaling is not specific only to erythroid lineages and has been confirmed in several solid tumors, including breast. Three different isoforms of erythropoietin receptor (EPOR) have been reported, the soluble (EPOR-S) and truncated (EPOR-T) forms acting antagonistically to the functional EPOR. In this study, we investigated the effect of human recombinant erythropoietin (rHuEPO) on cell proliferation, early gene response and the expression of EPOR isoforms in the MCF-7 breast cancer cell line. MATERIALS AND METHODS: The MCF-7 cells were cultured with or without rHuEPO for 72 h or 10 weeks and assessed for their growth characteristics, expression of early response genes and different EPOR isoforms. The expression profile of EPOR and EPOR-T was determined in a range of breast cancer cell lines and compared with their invasive properties. RESULTS: MCF-7 cell proliferation after rHuEPO treatment was dependent on the time of treatment and the concentration used. High rHuEPO concentrations (40 U/ml) stimulated cell proliferation independently of a preceding long-term exposure of MCF-7 cells to rHuEPO, while lower concentrations increased MCF-7 proliferation only after 10 weeks of treatment. Gene expression analysis showed activation of EGR1 and FOS, confirming the functionality of EPOR. rHuEPO treatment also slightly increased the expression of the functional EPOR isoform, which, however, persisted throughout the 10 weeks of treatment. The expression levels of EPOR-T were not influenced. There were no correlations between EPOR expression and the invasiveness of MCF-7, MDA-MB-231, Hs578T, Hs578Bst, SKBR3, T-47D and MCF-10A cell lines. CONCLUSIONS: rHuEPO modulates MCF-7 cell proliferation in time- and concentration-dependent manner. We confirmed EGR1, FOS and EPOR as transcription targets of the EPO-EPOR signaling loop, but could not correlate the expression of different EPOR isoforms with the invasiveness of breast cancer cell lines.

Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14alpha-demethylase (Cyp51) resembles Antley-Bixler syndrome.

Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.

Relationship between anatomical characteristics and personality traits in Lipizzan horses.

We tested 35 Lipizzan horses older than 5 years, ridden and healthy in three behavioural tests (handling, fear-reaction, and target training test). Physiological (heart rate and heart rate variability) and anatomical measurements (120 head and body distances and angles) were collected to validate parameters that reliably inform on handling/cooperation, fear/exploration and trainability in horses. Utilizing a standard clustering methodology on the behavioural data, we identified four general types of responses and categorised an individual as intermediate, low fearful, horses with low cooperation or low trainability. We additionally analysed the head morphology following Tellington-Jones and Taylor recommendations and correlated the measurements with data from a horse personality questionnaire. Although allocation to a particular personality group was not associated with these two methods, these groups differed in six anatomical characteristics of head and body. Regardless of the group, our results indicated that shorter horses (<75.9 cm) with a wider muzzle (>10.5 cm) are trustworthy, less fearful and easier to handle and train. We also demonstrated that horses with stronger legs and a wider base of the head have a lower heart rate when exposed to the second trial of the handling test.

Report on isoelectric focusing trial of erythropoietin profiling in two cancer patients during chemotherapy and darbepoetin treatment.

The level of erythropoietin, main regulator of erythropoiesis, is affected by hypoxia, anaemia, application of recombinant erythropoietin, chemotherapy and others. Isoelectric focusing (IEF) combined with double immunobloting is a method that enables distinct analysis of endogenous and recombinant erythropoietin isoforms. Aim of our study was to set up analysis of treatment effects on the pattern of endogenous erythropoietin in anaemic breast cancer patient. Urine and blood samples were collected during and after termination of the treatment and analysed by isoelectric focusing. Endogenous erythropoietin was found lower, but still detectable during darbepoetin treatment. Normal shift of erythropoietin isoforms between serum vs. urine, ordinary seen in healthy volunteers, was not observed indicating kidney damage. The patient was suffering from heavy proteinuria and had low Glomerular filtration rate indicating acute renal failure, probably caused by clinical status or cisplatin chemotherapy. IEF has not yet been used for follow up of erythropoietin profile in cancer patients. It enables to monitor the effects of treatment on the level of endogenous erythropoietin and indirectly indicates kidney function.

Molecular Pathways Involved in the Development of Congenital Erythrocytosis.

Patients with idiopathic erythrocytosis are directed to targeted genetic testing including nine genes involved in oxygen sensing pathway in kidneys, erythropoietin signal transduction in pre-erythrocytes and hemoglobin-oxygen affinity regulation in mature erythrocytes. However, in more than 60% of cases the genetic cause remains undiagnosed, suggesting that other genes and mechanisms must be involved in the disease development. This review aims to explore additional molecular mechanisms in recognized erythrocytosis pathways and propose new pathways associated with this rare hematological disorder. For this purpose, a comprehensive review of the literature was performed and different in silico tools were used. We identified genes involved in several mechanisms and molecular pathways, including mRNA transcriptional regulation, post-translational modifications, membrane transport, regulation of signal transduction, glucose metabolism and iron homeostasis, which have the potential to influence the main erythrocytosis-associated pathways. We provide valuable theoretical information for deeper insight into possible mechanisms of disease development. This information can be also helpful to improve the current diagnostic solutions for patients with idiopathic erythrocytosis.

Integration and Visualization of Regulatory Elements and Variations of the EPAS1 Gene in Human.

Endothelial PAS domain-containing protein 1 (EPAS1), also HIF2α, is an alpha subunit of hypoxia-inducible transcription factor (HIF), which mediates cellular and systemic response to hypoxia. EPAS1 has an important role in the transcription of many hypoxia-responsive genes, however, it has been less researched than HIF1α. The aim of this study was to integrate an increasing number of data on EPAS1 into a map of diverse OMICs elements. Publications, databases, and bioinformatics tools were examined, including Ensembl, MethPrimer, STRING, miRTarBase, COSMIC, and LOVD. The EPAS1 expression, stability, and activity are tightly regulated on several OMICs levels to maintain complex oxygen homeostasis. In the integrative EPAS1 map we included: 31 promoter-binding proteins, 13 interacting miRNAs and one lncRNA, and 16 post-translational modifications regulating EPAS1 protein abundance. EPAS1 has been associated with various cancer types and other diseases. The development of neuroendocrine tumors and erythrocytosis was shown to be associated with 11 somatic and 20 germline variants. The integrative map also includes 12 EPAS1 target genes and 27 interacting proteins. The study introduced the first integrative map of diverse genomics, transcriptomics, proteomics, regulomics, and interactomics data associated with EPAS1, to enable a better understanding of EPAS1 activity and regulation and support future research.

Congenital erythrocytosis - A condition behind recurrent thromboses: A case report and literature review.

Congenital erythrocytosis (CE) is an extremely rare disease and an infrequent cause of heamoglobin and haematocrit elevation. Genetic testing of CE is not widely available. Patients in whom a cause of erythrocytosis is not identified are classified as idiopathic erythrocytosis (IE) patients. In some types of CE thrombotic events have been reported but there is little hard evidence to advise on management in asymptomatic patients. Similarly is true for patients with IE. We describe a young patient who suffered several thromboembolic complications before the diagnosis of CE type 4 was established.

Erythrocytosis: genes and pathways involved in disease development.

Erythrocytosis is a blood disorder characterised by an increased red blood cell mass. The most common causes of erythrocytosis are acquired and caused by diseases and conditions that are accompanied by hypoxaemia or overproduction of erythropoietin. More rarely, erythrocytosis has a known genetic background, such as for polycythaemia vera and familial erythrocytosis. The majority of cases of polycythaemia vera are associated with acquired variants in JAK2, while familial erythrocytosis is a group of congenital disorders. Familial erythrocytosis type 1 is associated with hypersensitivity to erythropoietin (variants in EPOR), types 2-5 with defects in oxygen-sensing pathways (variants in VHL, EGLN1, EPAS1, EPO), and types 6-8 with an increased affinity of haemoglobin for oxygen (variants in HBB, HBA1, HBA2, BPGM). Due to a heterogenic genetic background, the causes of disease are not fully discovered and in more than 70% of patients the condition remains labelled idiopathic.The transfer of next-generation sequencing into clinical practice is becoming a reality enabling detection of various variants in a single rapid test. In this review, we describe the current research on erythrocytosis gene variants and the mechanisms associated with disease development, along with the currently used diagnostic tests.

Is It Possible to Predict Clonal Thrombocytosis in Triple-Negative Patients with Isolated Thrombocytosis Based Only on Clinical or Blood Findings?

JAK2, MPL, and CALR mutations define clonal thrombocytosis in about 90% of patients with sustained isolated thrombocytosis. In the remainder of patients (triple-negative patients) diagnosing clonal thrombocytosis is especially difficult due to the different underlying conditions and possible inconclusive bone marrow biopsy results. The ability to predict patients with sustained isolated thrombocytosis with a potential clonal origin has a prognostic value and warrants further examination. The aim of our study was to define a non-invasive clinical or blood parameter that could help predict clonal thrombocytosis in triple-negative patients. We studied 237 JAK2 V617-negative patients who were diagnosed with isolated thrombocytosis and referred to the haematology service. Sixteen routine clinical and blood parameters were included in the logistic regression model which was used to predict the type of thrombocytosis (reactive/clonal). Platelet count and lactate dehydrogenase (LDH) were the only statistically significant predictors of clonal thrombocytosis. The platelet count threshold for the most accurate prediction of clonal or reactive thrombocytosis was 449 × 109/L. Other tested clinical and blood parameters were not statistically significant predictors of clonal thrombocytosis. The level of LDH was significantly higher in CALR-positive patients compared to CALR-negative patients. We did not identify any new clinical or blood parameters that could distinguish clonal from reactive thrombocytosis. When diagnosing clonal thrombocytosis triple-negative patients are most likely to be misdiagnosed. Treatment in patients with suspected triple negative clonal thrombocytosis should not be delayed if cardiovascular risk factors or pregnancy coexist, even in the absence of firm diagnostic criteria. In those cases the approach "better treat more than less" should be followed.