Recently, an antibody which inhibits the glycoprotein A repetitions predominant (GARP)-mediated release of active transforming growth factor beta (TGFβ) from the TGFβ propeptide latency-associated peptide (LAP) showed preclinical activity in a murine model of the chronic myeloproliferative neoplasms (MPN). Consequently, we investigated the expression of the immunosuppressive molecules LAP and GARP on peripheral blood lymphocytes from 56 MPN patients and 11 healthy donors (HD). We found that lymphocytes from patients with MPN express higher levels of LAP and GARP with no strong differences found between the different MPN diagnoses. The impact of clinical parameters on the expression of LAP and GARP by lymphocytes showed that patients with calreticulin (CALR)mut MPN have increased expression compared with HD and patients with the Januskinase2 (JAK2) mutation. The fraction of lymphocytes bound to activated platelets (aPLT) strongly correlate to LAP and GARP expression suggesting that it is not the lymphocytes themselves but aPLT, which confer the increased expression of GARP and LAP on MPN patient lymphocytes. Notably, no differences in neither platelet counts nor anti-thrombotic therapy was identified between patients with JAK2- and CALRmut patients. Analysis of platelet gene expression failed to identify differences in expression of relevant genes between JAK2- and CALRmut patients.

Myelofibrosis (MF) is a clonal disorder of hematopoietic stem cells characterized by altered bone marrow function and fibrosis. The aim of this narrative review is to report on the most recent epidemiologic data and to discuss features of MF and current strategies for the management of this condition in clinical practice. MF features covered by our review will include: characteristics of patients with MF; myeloproliferative and myelodepletive phenotypes; MF-associated thrombosis and bleeding; risk of infections; prefibrotic and overt PMF; secondary MF. Finally, we will discuss a few aspects of MF management in clinical practice and suggest strategies for its optimization and standardization. The focus of our paper is on Italy, but relevant data from other countries will also be reviewed.

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Circular RNAs (circRNAs) are covalently closed, single-stranded RNAs that play critical roles in various biological processes and diseases, including cancers. However, the functions and mechanisms of circRNAs in hepatocellular carcinoma (HCC) need further clarification. Here, we identified and confirmed that circATF6 is downregulated in HCC tissues and negatively associated with the overall survival of HCC patients. Ectopic overexpression of circATF6 inhibits malignant phenotypes of HCC cells in vitro and in vivo, while knockdown of circATF6 had opposite effects. Mechanistically, we found that circATF6 bound to calreticulin (CALR) protein and acted as a scaffold to enhance the interaction of CALR with calpain2 (CAPN2), which promoted the degradation of CALR by its enzymatic activity. Moreover, we found that circATF6 inhibited HCC cells by suppressing CALR-mediated wnt/β-catenin signaling pathway. Taken together, our findings suggest that circATF6 is a potential prognostic biomarker and therapeutic target for HCC.

We previously reported the Marimo cell line, which was established from the bone marrow cells of a patient with essential thrombocythemia (ET) at the last stage after transformation to acute myeloid leukemia (AML). This cell line is widely used for the biological analysis of ET because it harbors CALR mutation. However, genetic processes during disease progression in the original patient were not analyzed. We sequentially analyzed the genetic status in the original patient samples during disease progression. The ET clone had already acquired CALR and MPL mutations, and TP53 and NRAS mutations affected the disease progression from ET to AML in this patient. Particularly, the variant allele frequency of the NRAS mutation increased along with the disease progression after transformation, and the NRAS-mutated clone selectively proliferated in vitro, resulting in the establishment of the Marimo cell line. Although CALR and MPL mutations co-existed, MPL was not expressed in Marimo cells or any clinical samples. Furthermore, mitogen-activated protein kinase (MAPK) but not the JAK2-STAT pathway was activated. These results collectively indicate that MAPK activation is mainly associated with the proliferation ability of Marimo cells.

Essential thrombocythemia (ET) is a blood cancer caused by mutations in JAK2 and CALR. It is widely recognized that both mutations lead to the constitutive activation of JAK2/STAT signaling, although other JAK/STAT-independent pathogenic mechanisms triggered by these alterations have also been described in ET. In an attempt to study JAK2/STAT-independent mechanisms derived from CALR mutations, our research group created a C. elegans model with patient-like mutations in calreticulin that lacks JAK counterparts. The introduction of patient-like mutations in the calreticulin of C. elegans leads to an increase in the transcriptional expression of nhr-2, independently of JAK2/STAT activation. In the present study, we aim to verify if this mechanism is conserved in patients with ET harboring CALR mutations. To do so, we evaluated the expression of potential orthologs of nhr-2 in human cell lines of interest for the study, as well as in bone marrow (BM) or peripheral blood (PB) mononuclear cells from patients with CALR or JAK2 mutations. The results revealed that this mechanism is conserved in CALR-mutated ET patients, since CALR, but not JAK2 mutations, were associated with an overexpression of RXRA in patients with ET. The use of drugs targeting the activation or blockade of this target in the analyzed cell lines did not result in changes in cell viability. However, RXRA might be relevant in the disease, pointing to the need for future research testing retinoids and other drugs targeting RXRα for the treatment of ET patients.

Paroxysmal nocturnal hemoglobinuria (PNH) results from the loss of erythrocyte surface proteins, leading to complement activation and its spectrum of effects. We explore this case of a 57-year-old man with post-essential thrombocythemia (ET) myelofibrosis (MF) who developed symptomatic anemia with evidence of hemolysis on lab work. While hemolysis was localized to be intramedullary based on workup, the exact diagnosis was undetermined, leading to a prolonged course of steroid therapy to control anemia. The hemolysis was eventually attributed to PNH diagnosed on flow cytometry and the patient was treated with complement inhibitors with eventual failure of therapy. He ultimately underwent a successful hematopoietic cell transplant (HCT) with post-transplantation flow cytometry showing complete resolution of PNH. While PNH has been identified as a progression of myelodysplastic syndromes, the mechanisms of its rare development in myeloproliferative neoplasms are poorly elucidated. Furthermore, its rarity and often vague symptoms make diagnosis and treatment a challenge. This is the second reported case of a JAK2-negative, CALR-positive post-ET MF and the first reported case to be treated with HCT. This case probes for further insight into the clinical significance between MF and PNH, its impact on management, and further consideration for HCT as curative therapy in such patients who fail complement inhibitor therapy.

Myeloproliferative neoplasms (MPNs) are subdivided into Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) and Ph-negative MPNs. BCR::ABL1 translocation is essential for the development and diagnosis of CML; on the other hand, the majority of Ph-negative MPNs are characterized by generally mutually exclusive mutations of Janus kinase 2 (JAK2), calreticulin (CALR), or thrombopoietin receptor/myeloproliferative leukemia (MPL). CALR mutations have been described essentially in JAK2 and MPL wild-type essential thrombocythemia and primary myelofibrosis. Rarely coexisting CALR and MPL mutations have been found in Ph-negative MPNs. BCR::ABL1 translocation and JAK2 mutations were initially considered mutually exclusive genomic events, but a discrete number of cases with the combination of these genetic alterations have been reported. The presence of BCR::ABL1 translocation with a coexisting CALR mutation is even more uncommon. Herein, starting from a routinely diagnosed case of CALR-mutated primary myelofibrosis subsequently acquiring BCR::ABL1 translocation, we performed a comprehensive review of the literature, discussing the clinicopathologic and molecular features, as well as the outcome and treatment of cases with BCR::ABL1 and CALR co-occurrence.

This study aims to investigate the role of anoikis-related genes in diabetic foot (DF) by utilizing bioinformatics analysis to identify key genes associated with anoikis in DF. We selected the GEO datasets GSE7014, GSE80178 and GSE68183 for the extraction and analysis of differentially expressed anoikis-related genes (DE-ARGs). GO analysis and KEGG analysis indicated that DE-ARGs in DF were primarily enriched in apoptosis, positive regulation of MAPK cascade, anoikis, focal adhesion and the PI3K-Akt signalling pathway. Based on the LASSO and SVM-RFE algorithms, we identified six characteristic genes. ROC curve analysis revealed that these six characteristic genes had an area under the curve (AUC) greater than 0.7, indicating good diagnostic efficacy. Expression analysis in the validation set revealed downregulation of CALR in DF, consistent with the training set results. GSEA results demonstrated that CALR was mainly enriched in blood vessel morphogenesis, endothelial cell migration, ECM-receptor interaction and focal adhesion. The HPA database revealed that CALR was moderately enriched in endothelial cells, and CALR was found to interact with 63 protein-coding genes. Functional analysis with DAVID suggested that CALR and associated genes were enriched in the phagosome component. CALR shows promise as a potential marker for the development and treatment of DF.

Myeloproliferative neoplasms (MPNs) are hematopoietic diseases characterized by the clonal expansion of single or multiple lineages of differentiated myeloid cells that accumulate in the blood and bone marrow. MPNs are grouped into distinct categories based on key clinical presentations and distinctive mutational hallmarks. These include chronic myeloid leukemia (CML), which is strongly associated with the signature BCR::ABL1 gene translocation, polycythemia vera (PV), essential thrombocythemia (ET), and primary (idiopathic) myelofibrosis (PMF), typically accompanied by molecular alterations in the JAK2, MPL, or CALR genes. There are also rarer forms such as chronic neutrophilic leukemia (CNL), which involves mutations in the CSF3R gene. However, rather than focusing on the differences between these alternate disease categories, this review aims to present a unifying molecular etiology in which these overlapping diseases are best understood as disruptions of normal hematopoietic signaling: specifically, the chronic activation of signaling pathways, particularly involving signal transducer and activator of transcription (STAT) transcription factors, most notably STAT5B, leading to the sustained stimulation of myelopoiesis, which underpins the various disease sequalae.