Characterization of Philadelphia-negative Chronic Myeloproliferative Neoplasms: identification of novel biomarkers by Next Generation Sequencing and study of interactions between hematopoietic stem cell and the inflammatory cell micro-environment

Myeloproliferative Neoplasms (MPN) are a heterogeneous group of hematological malignancies, consisting in clonal disorders of the hematopoietic stem/progenitor cell (HSC/PC). Molecular alterations and inflammatory microenvironment represent the two main etiopathogenic factors of MPN. The aim of this study was the molecular characterization of MPN patients and the study of interactions between HSC/PC and the inflammatory cell micro-environment. We investigated young (<40 years at diagnosis) ET and early-PMF patients, and patients with a JAK2V617F allele burden (AB) <3%, demonstrating that its determination is relevant both at diagnosis and during follow up. Indeed, it allows to prove the presence of a clonal hematopoiesis and, also, to predict clinical outcome. Of note, an AB≥0,8% always corresponds to an overt MPN phenotype. In this context, coordinating a network of 19 Italian laboratories, we identified the ipsogen JAK2 MutaQuant kit as the most sensitive and efficient assay for the quantification of samples with different mutation loads (in particular those with AB≤1%). With regard to the role of inflammatory microenvironment in the pathogenesis of MF, IL-1β and TIMP-1 seemed to confer a survival advantage to MF-derived HSPCs, enhancing their proliferation and in vitro migration, as well as their clonogenic ability. Finally, in this study we tested three different gene panels for mutations detection, obtaining promising results in terms of coverage analysis (more than 95% of target regions with depth greater than 500X) and identifying gene variants with very low mutation load (<1%) in all patients. In conclusion, this study set the basis for the standardization of molecular techniques for the determination of JAK2V617F AB, and for the validation of a robust NGS approach to be translated into a diagnostic setting. Moreover, IL-1β and TIMP-1 emerged as novel promoting factors of the in vitro maintenance of MF-derived HSPC, which may be exploited as potential targets of therapy

A tool to observe the phenomenology and aesthetics of primary relationships: the “dance steps” of reciprocity between caregivers and infant/child – Pilot validity study

This study addresses the complexity of caregiver-infant/child interactions from the theoretical frame of Gestalt psychotherapy and the field of application of Pediatric Psychology. Based on a previous empirical study on the process of reciprocity in caregiver-infant/child interactions (Spagnuolo Lobb, 2016), the authors have worked on the construction of an observational tool to look at the co-creation of meaningful experiences, switching the focus from the child to the “dance” of reciprocity between caregiver and infant/child. Considering the contextualization in the field of Pediatric Psychology, this pilot study aimed to test the tool’s application with caregiverpreterm infant dyads, but exclusively referring to moderately preterm birth condition and with lack of disability or in any case serious evolutionary compromises. The study measured: 1. the content/construct validity of the tool, 2. its internal reliability, 3. its “sensitivity” to grasp the changing of the relational “dance” in the transition from one developmental step of the infant to another, regarding the times considered (between 6-9-12 months of corrected age), 4. co-occurrences between the behavioral flows of the infant-caregiver dyad in the 3 developmental stages considered. 32 expert psychotherapists were involved in measuring the instrument’s validity, and 13 caregiver-infant dyads were observed in their interactions at 6-9-12 months of corrected age of the infant. This pilot study promises to define – both for preventative and psychotherapeutic interventions – specific parental competencies for the infant’s neurodevelopment in the first year of lif

Circulating calreticulin is increased in myelofibrosis: Correlation with interleukin-6 plasma levels, bone marrow fibrosis, and splenomegaly

Myelofibrosis (MF) is a clonal neoplasia of the hemopoietic stem/progenitor cells associated with genetic mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) genes. MF is also characterized by a state of chronic inflammation. Calreticulin (CRT), as a multifunctional protein, is involved in a spectrum of cellular processes including inflammation, autoimmunity, and cancer initiation/progression. Based on this background, we hypothesised that in MF circulating CRT might reflect the inflammatory process. In the present study we show that circulating CRT is increased in MF patients compared to healthy controls. Also, in MF, CRT levels highly correlate with bone marrow fibrosis, splenomegaly, and Interleukin-6 (IL-6) plasma levels. In turn, higher IL-6 levels also correlated with disease severity in terms of increased spleen size, bone marrow fibrosis, number of circulating CD34+ cells, and lower hemoglobin values. These results demonstrate that the circulating CRT takes part in the inflammatory network of MF and correlates with aggressiveness of the disease

Circulating Calreticulin Is Increased in Myelofibrosis: Correlation with Interleukin-6 Plasma Levels, Bone Marrow Fibrosis, and Splenomegaly

Myelofibrosis (MF) is a clonal neoplasia of the hemopoietic stem/progenitor cells associated with genetic mutations in the Janus kinase 2 (JAK2), myeloproliferative leukemia virus oncogene (MPL), and calreticulin (CALR) genes. MF is also characterized by a state of chronic inflammation. Calreticulin (CRT), as a multifunctional protein, is involved in a spectrum of cellular processes including inflammation, autoimmunity, and cancer initiation/progression. Based on this background, we hypothesised that in MF circulating CRT might reflect the inflammatory process. In the present study we show that circulating CRT is increased in MF patients compared to healthy controls. Also, in MF, CRT levels highly correlate with bone marrow fibrosis, splenomegaly, and Interleukin-6 (IL-6) plasma levels. In turn, higher IL-6 levels also correlated with disease severity in terms of increased spleen size, bone marrow fibrosis, number of circulating CD34+ cells, and lower hemoglobin values. These results demonstrate that the circulating CRT takes part in the inflammatory network of MF and correlates with aggressiveness of the disease

Subclinical synovitis detected by ultrasound in children affected by coeliac disease: a frequent manifestation improved by a gluten-free diet

Objective Coeliac disease (CD) is a chronic autoimmune disease of the small intestine caused by the ingestion of gluten, in which musculoskeletal manifestations may occur. Aim of this study was to evaluate the prevalence and severity of joint involvement in paediatric patients with CD using musculoskeletal ultrasound (US). Methods Consecutive paediatric CD patients were enrolled and underwent US evaluations at level of knees, hips and ankles. The presence of joint effusion (JE), synovial hypertrophy, power Doppler signal and structural damage lesions (bone irregularities and erosions) was registered. Inflammatory abnormalities were scored on a semi-quantitative scale (0-3), and structural damage lesions on a dichotomous scale (0-1). Results Seventy-four CD children (mean age: 7.6 years; range: 1-14.2; M/F 24150) were enrolled. Thirty-eight were on a gluten-containing diet (GCD) and 36 on a gluten-free diet (GFD). US showed the presence of abnormalities in 23 patients overall (31.1%); JE was the most frequently observed change (23123). US abnormalities were observed in 19 patients (50.0%) of GCD group and in 4 of GFD group (11.1%, p=0.007). Interestingly, 12123 (52.2%) patients with US-detected changes were asymptomatic. Conclusion This is the first US study demonstrating joint involvement in children with CD. JE, the most frequent manifestation, was present also in asymptomatic patients and was reduced in those on GFD. These findings may indicate that, also at joint level, an inflammatory response represented by the appearance of TE may be induced by exposure to gluten

Crucial factors of the inflammatory microenvironment (IL-1\u3b2/ TNF-\u3b1/TIMP-1) promote the maintenance of the malignant hemopoietic clone of myelofibrosis: an in vitro study

Along with molecular abnormalities (mutations in JAK2, Calreticulin (CALR) and MPL genes), chronic inflammation is the major hallmark of Myelofibrosis (MF). Here, we investigated the in vitro effects of crucial factors of the inflammatory microenvironment (Interleukin (IL)-1\u3b2, Tumor Necrosis Factor (TNF)-\u3b1, Tissue Inhibitor of Metalloproteinases (TIMP)-1 and ATP) on the functional behaviour of MF-derived circulating CD34+ cells.We found that, regardless mutation status, IL-1\u3b2 or TNF-\u3b1 increases the survival of MF-derived CD34+ cells. In addition, along with stimulation of cell cycle progression to the S-phase, IL-1\u3b2 or TNF-\u3b1 \ub1 TIMP-1 significantly stimulate(s) the in vitro clonogenic ability of CD34+ cells from JAK2V617 mutated patients. Whereas in the JAK2V617F mutated group, the addition of IL-1\u3b2 or TNF-\u3b1 + TIMP-1 decreased the erythroid compartment of the CALR mutated patients. Megakaryocyte progenitors were stimulated by IL-1\u3b2 (JAK2V617F mutated patients only) and inhibited by TNF-\u3b1. IL-1\u3b2 + TNF-\u3b1 + C-X-C motif chemokine 12 (CXCL12) \ub1 TIMP-1 highly stimulates the in vitro migration of MF-derived CD34+ cells. Interestingly, after migration toward IL-1\u3b2 + TNF-\u3b1 + CXCL12 \ub1 TIMP-1, CD34+ cells from JAK2V617F mutated patients show increased clonogenic ability.Here we demonstrate that the interplay of these inflammatory factors promotes and selects the circulating MF-derived CD34+ cells with higher proliferative activity, clonogenic potential and migration ability. Targeting these micro-environmental interactions may be a clinically relevant approach

Mutations in JAK2 and Calreticulin genes are associated with specific alterations of the immune system in myelofibrosis

Myelofibrosis (MF) is a clonal neoplasia associated with chronic inflammation due to aberrant cytokine production. Mutations in Janus Kinase-2 (JAK2), calreticulin (CALR) and myeloproliferative leukemia protein (MPL) genes have been recently associated to MF and they all activate the JAK/STAT signaling pathway. Since this pathway is essential in shaping the immune response, we investigated the role of circulating immune subsets and cytokines in 38 patients (20 carrying JAK2(V617F),13 exon-9 CALR mutation and 5 triple negative). In comparison to healthy donors, patients presented a reduced amount of circulating dendritic cells (DCs) associated with a defective ability of monocytes in differentiating into DCs. In addition, we found a reduction in circulating T-helper (Th)1 and Th17 and hypo-functional innate lymphoid cells (ILC). Results analyzed according to the mutational status showed that patients carrying JAK2(V617F) mutation had a reduction in Th17, myeloid-DCs and effector Tregs as well as increased ILC1 and cytokine producing Tregs. The CALR mutated patients revealed high ILC3 levels, reduced Th1 and their monocytes had a reduced capacity to mature in vitro into fully committed DCs. Their Tregs were also less effective in inhibiting the proliferation of autologous effector T-cells due to an increased proliferative status induced by CALR mutation. Triple negative patients presented a reduced amount of total circulating CD3, effectors Tregs and Th1 with increased ILC1. Overall, we have demonstrated that in MF different mutations lead to phenotypic and functional alterations in different immune subsets that may have a potential role in disease progression and susceptibility to infections

The relevance of a low JAK2V617F allele burden in clinical practice: a monocentric study

Since low JAK2V617F allele burden (AB) has been detected also in healthy subjects, its clinical interpretation may be challenging in patients with chronic myeloproliferative neoplasms (MPNs). We tested 1087 subjects for JAK2V617F mutation on suspicion of hematological malignancy. Only 497 (45.7%) patients were positive. Here we present clinical and laboratory parameters of a cohort of 35/497 patients with an AB ≤ 3%.Overall, 22/35 (62.9%) received a WHO-defined diagnosis of MPN and in 14/35 cases (40%) diagnosis was supported by bone marrow (BM) histology (''Histology-based'' diagnosis). In patients that were unable or refused to perform BM evaluation, diagnosis relied on prospective clinical observation (12 cases, 34.3%) and molecular monitoring (6 cases, 17.1%) (''Clinical-based'' or ''Molecular-based'' diagnosis, respectively). In 11/35 (31.4%) patients, a low JAK2V617F AB was not conclusive of MPN. The probability to have a final hematological diagnosis (ET/PV/MF) was higher in patients with thrombocytosis than in patients with polyglobulia (73.7% vs 57.1%, respectively). The detection of AB ≥ 0.8% always corresponded to an overt MPN phenotype. The repetition of JAK2V617F evaluation over time timely detected the spontaneous expansion (11 cases) or reduction (4 cases) of JAK2V617F-positive clones and significantly oriented the diagnostic process.Our study confirms that histology is relevant to discriminate small foci of clonal hematopoiesis with uncertain clinical significance from a full blown disease. Remarkably, our data suggest that a cut-off of AB ≥ 0.8% is very indicative for the presence of a MPN. Monitoring of the AB over time emerged as a convenient and non-invasive method to assess clonal hematopoiesis expansion