Calreticulin (CALR) is a chaperone present in the endoplasmic reticulum, which is involved in the quality control of N-glycosylated proteins and storage of calcium ions. In 2013, the C-terminal ...mutation in CALR was identified in half of the patients with essential thrombocythemia and primary myelofibrosis who did not have a JAK2 or MPL mutation. The results of 8 years of intensive research are changing the clinical practice associated with treating myeloproliferative neoplasms (MPNs). The presence or absence of CALR mutations and their mutation types already provide important information for diagnosis and treatment decision making. In addition, the interaction with the thrombopoietin receptor MPL, which is the main mechanism of transformation by CALR mutation, and the expression of the mutant protein on the cell surface have a great potential as targets for molecular-targeted drugs and immunotherapy. This chapter presents recent findings on the clinical significance of the CALR mutation and the molecular basis by which this mutation drives MPNs.
High frequency of
JAK2
V617F or
CALR
exon 9 mutations is a main molecular feature of myeloproliferative neoplasms (MPNs). Analysis of mouse models driven by these mutations suggests that they are a ...direct cause of MPNs and that the expression levels of the mutated genes define the disease phenotype. The function of MPN-initiating cells has also been elucidated by these mouse models. Such mouse models also play an important role in modeling disease to investigate the effects and action mechanisms of therapeutic drugs, such as JAK2 inhibitors and interferon α, against MPNs. The mutation landscape of hematological tumors has already been clarified by next-generation sequencing technology, and the importance of functional analysis of mutant genes in vivo should increase further in the future.
Since the mouse models with the driver mutations of myeloproliferative neoplasms (MPNs) reproduce various pathological conditions found in MPNs patients such as blood cell increase, splenomegaly, and ...bone marrow fi brosis, these mouse models are extremely useful as tools to elucidate the mechanism of pathogenesis. Activation of MPL and its downstream signaling pathways is common to the JAK2, MPL, and CALR mutations, which are major driver mutations in MPNs, and is essential for the pathogenesis of MPNs. We analyzed the gene expression profi le of MPLexpressing cells in Jak2V617F transgenic mice and identified the signaling pathway responsible for bone marrow fi brosis. MPL activates the transcription factor upstream transcription factor via the p42/44 MAPK pathway, and the JAK2V617F mutant protein uses this pathway to enhance transcription of TGF-β1, a cytokine essential for bone marrow fibrosis. Inhibition of this pathway effectively suppressed organ fibrosis in Jak2V617F mice in vivo. JAK2 inhibitors have only a slow and limited effect on improving myelofibrosis. The mechanism we have discovered is a promising therapeutic target for antifi brotic therapy, which has recently been developed for patients with myelofi brosis. Such anti-fi brotic therapies may create new therapeutic benefi ts for patients with myelofi brosis that cannot be provided by JAK2 inhibitors. Since the mouse models with the driver mutations of myeloproliferative neoplasms (MPNs) reproduce various pathological conditions found in MPNs patients such as blood cell increase, splenomegaly, and bone marrow fi brosis, these mouse models are extremely useful as tools to elucidate the mechanism of pathogenesis. Activation of MPL and its downstream signaling pathways is common to the JAK2, MPL, and CALR mutations, which are major driver mutations in MPNs, and is essential for the pathogenesis of MPNs. We analyzed the gene expression profi le of MPLexpressing cells in Jak2V617F transgenic mice and identified the signaling pathway responsible for bone marrow fi brosis. MPL activates the transcription factor upstream transcription factor via the p42/44 MAPK pathway, and the JAK2V617F mutant protein uses this pathway to enhance transcription of TGF-β1, a cytokine essential for bone marrow fibrosis. Inhibition of this pathway effectively suppressed organ fibrosis in Jak2V617F mice in vivo. JAK2 inhibitors have only a slow and limited effect on improving myelofibrosis. The mechanism we have discovered is a promising therapeutic target for antifi brotic therapy, which has recently been developed for patients with myelofi brosis.Such anti-fi brotic therapies may create new therapeutic benefi ts for patients with myelofi brosis that cannot be provided by JAK2 inhibitors.
Pulmonary hypertension (PH) is a progressive cardiopulmonary disease characterized by pulmonary arterial remodeling. Clonal somatic mutations including JAK2V617F, the most frequent driver mutation ...among myeloproliferative neoplasms, have recently been identified in healthy individuals without hematological disorders. Here, we reveal that clonal hematopoiesis with JAK2V617F exacerbates PH and pulmonary arterial remodeling in mice. JAK2V617F-expressing neutrophils specifically accumulate in pulmonary arterial regions, accompanied by increases in neutrophil-derived elastase activity and chemokines in chronic hypoxia-exposed JAK2V617F transgenic (JAK2
) mice, as well as recipient mice transplanted with JAK2
bone marrow cells. JAK2V617F progressively upregulates Acvrl1 (encoding ALK1) during the differentiation from bone marrow stem/progenitor cells peripherally into mature neutrophils of pulmonary arterial regions. JAK2V617F-mediated STAT3 phosphorylation upregulates ALK1-Smad1/5/8 signaling. ALK1/2 inhibition completely prevents the development of PH in JAK2
mice. Finally, our prospective clinical study identified JAK2V617F-positive clonal hematopoiesis is more common in PH patients than in healthy subjects. These findings indicate that clonal hematopoiesis with JAK2V617F causally leads to PH development associated with ALK1 upregulation.
The aim of this study was to elucidate the role of a non-canonical
JAK2
mutation
JAK2
-T875N, which was identified by exome sequencing in a patient with essential thrombocytosis (ET) who had a family ...history of suspecting ET. Whole exome sequencing was performed on peripheral blood mononuclear cells and buccal swab-derived genomic DNA. Sanger sequencing was performed to confirm the variant. We evaluated the function of the mutation on JAK2 activity and downstream signaling (Erk, STATs) using JAK2-T875N-transfected or transduced cell lines. 293T cells transfected with
JAK2
cDNA carrying V617F or T875N mutations showed increased levels of phosphorylated JAK2 and Erk. Enhanced STAT3 and STAT5 activity was confirmed by promoter assay.
JAK2
-T875N-transduced Ba/F3 cells showed increased cellular growth without IL-3 stimulation. To our knowledge, this is the first case of ET caused by
JAK2
-T875N mutation with a family history of thrombocytosis and cerebral infarction.
Although splenomegaly is one of the important signs of primary myelofibrosis, the differential diagnosis varies from malignant disorders to benign disorders, including malignant lymphoma and ...sarcoidosis. The patient was a 67-year-old male who developed anemia and huge splenomegaly. The laboratory findings include human T-cell leukemia virus type 1 (HTLV-1) antibody, elevated soluble interleukin-2 receptor, hypocellular bone marrow, and uptake in the spleen on positron emission tomography/computed tomography scan. Additionally, we performed laparoscopic splenectomy to alleviate the clinical symptoms and to rule out malignant lymphoma. Histological findings revealed extramedullary hematopoiesis, characterized by the presence of erythroid islands and clusters of dysplastic megakaryocytes with increased reticulin fibrosis. Immunohistochemical staining revealed the presence of von Willebrand factor, dysplastic megakaryocytes, myeloperoxidase, myeloid-predominant proliferations, and CD34 immature myeloid cells. Furthermore, regarding the angiogenesis in the spleen, the endothelial cells of the capillaries and those of the sinusoidal vascular system that were reactive for CD34 and CD8, respectively, were also detected. Consequently, the histological findings revealed both extramedullary hematopoiesis and angiogenesis in spleen. Based on the histological findings and the identification of Janus activating kinase 2 (JAK-2) mutation, the patient was diagnosed with primary myelofibrosis. Splenectomy reduces blood transfusion requirements after surgery. The patient was carefully followed-up without further treatments. Thus, primary myelofibrosis is the crucial differential diagnosis of huge splenomegaly.
Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) characterized by clonal myeloproliferation, progressive bone marrow (BM) fibrosis, splenomegaly, and anemia. BM fibrosis was ...previously thought to be a reactive phenomenon induced by mesenchymal stromal cells that are stimulated by the overproduction of cytokines such as transforming growth factor (TGF)-β1. However, the involvement of neoplastic fibrocytes in BM fibrosis was recently reported. In this study, we showed that the vast majority of collagen- and fibronectin-producing cells in the BM and spleens of Jak2V617F-induced myelofibrosis (MF) mice were fibrocytes derived from neoplastic hematopoietic cells. Neoplastic monocyte depletion eliminated collagen- and fibronectin-producing fibrocytes in BM and spleen, and ameliorated most characteristic MF features in Jak2V617F transgenic mice, including BM fibrosis, anemia, and splenomegaly, while had little effect on the elevated numbers of megakaryocytes and stem cells in BM, and leukothrombocytosis in peripheral blood. TGF-β1, which was produced by hematopoietic cells including fibrocytes, promoted the differentiation of neoplastic monocytes to fibrocytes, and elevated plasma TGF-β1 levels were normalized by monocyte depletion. Collectively, our data suggest that neoplastic fibrocytes are the major contributor to BM fibrosis in PMF, and TGF-β1 is required for their differentiation.
The occurrence of a mediastinal germ cell tumor (GCT) and hematological malignancy in the same patient is very rare. Due to its rarity, there have been only two reports of the concurrent cases ...undergoing detailed genetic analysis with whole-exome sequencing (WES), and the possible clonal relationship between the both tumors remained not fully elucidated.
We performed whole-exome sequencing analysis of mediastinal GCT and acute myeloid leukemia (AML) samples obtained from one young Japanese male adult patient with concurrent both tumors, and investigated the possible clonal relationship between them.
Sixteen somatic mutations were detected in the mediastinal GCT sample and 18 somatic mutations in the AML sample. Mutations in nine genes, including TP53 and PTEN both known as tumor suppressor genes, were shared in both tumors.
All in our case and in the previous two cases with concurrent mediastinal GCT and AML undergoing with whole-exome sequencing analysis, TP53 and PTEN mutations were commonly shared in both tumors. These data not only suggest that these tumors share a common founding clone, but also indicate that associated mediastinal GCT and AML harboring TP53 and PTEN mutations represent a unique biological entity.
Whether human T-lymphotropic virus type 1 (HTLV-1) carriers can develop sufficient humoral immunity after coronavirus disease 2019 (COVID-19) vaccination is unknown.
To investigate humoral immunity ...after COVID-19 vaccination in HTLV-1 carriers, a multicenter, prospective observational cohort study was conducted at five institutions in southwestern Japan, an endemic area for HTLV-1. HTLV-1 carriers and HTLV-1-negative controls were enrolled for this study from January to December 2022. During this period, the third dose of the COVID-19 vaccine was actively administered. HTLV-1 carriers were enrolled during outpatient visits, while HTLV-1-negative controls included health care workers and patients treated by participating institutions for diabetes, hypertension, or dyslipidemia. The main outcome was the effect of HTLV-1 infection on the plasma anti-COVID-19 spike IgG (IgG-S) titers after the third dose, assessed by multivariate linear regression with other clinical factors.
We analyzed 181 cases (90 HTLV-1 carriers, 91 HTLV-1-negative controls) after receiving the third dose. HTLV-1 carriers were older (median age 67.0 vs. 45.0 years, p < 0.001) and more frequently had diabetes, hypertension, or dyslipidemia than did HTLV-1-negative controls (60.0% vs. 27.5%, p < 0.001). After the third dose, the IgG-S titers decreased over time in both carriers and controls. Multivariate linear regression in the entire cohort showed that time since the third dose, age, and HTLV-1 infection negatively influenced IgG-S titers. After adjusting for confounders such as age, or presence of diabetes, hypertension, or dyslipidemia between carriers and controls using the overlap weighting propensity score method, and performing weighted regression analysis in the entire cohort, both time since the third dose and HTLV-1 infection negatively influenced IgG-S titers.
The humoral immunity after the third vaccination dose is impaired in HTLV-1 carriers; thus, customized vaccination schedules may be necessary for them.
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