Myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). MPN are characterized by clonal proliferation of myeloid progenitors leading to erythrocytosis, thrombocytosis, or leukocytosis, and risk of hemorrhagic and thrombotic events, as well as myelofibrosis and blast transformation. The discovery of somatic mutations in MPN, namely JAK2 V617F, JAK2 exon 12, MPL, and CALR mutations, has permitted a more specific approach to diagnosis and treatment. The prevalence of JAK2 V617F mutations is higher than 95% in PV, 50%-75% in ET and 40%-75% in PMF. JAK2 exon 12 mutations are specific of PV. A 20%-30% of patients with ET and PMF present a CALR mutation. The screening of mutations strengthens the diagnosis of MPN since 97% of MPN have at least 1 somatic mutation. Interestingly, different mutations grant different phenotype and prognosis. Of particular importance, CALR mutations grant a favorable prognosis in ET and PMF, while ASXL1 mutations confer a poorer outcome. In fact, the use of CALR/ASXL1 status for the prognostication of patients has increased clinical value and is now suggested for guidance of therapy in PMF. The increasing importance of mutations in the management of MPN warrants a more frequent revision of current diagnostic criteria and prognostic models and a better understanding of the mechanisms leading to MPN subset differentiation.

To investigate the current situation and issues regarding the diagnosis of Philadelphia-negative myeloproliferative neoplasms (MPN) in Japan, we retrospectively analyzed an accumulated cohort consisting of 1,081 patients with suspected MPN. Based on WHO2008 diagnostic criteria, we diagnosed 101 of these patients with polycythemia vera, 179 with essential thrombocythemia, 36 with primary myelofibrosis, 45 with unclassifiable MPN, and 4 with myelodysplastic syndromes. Out of 716 patients, 235 were not diagnosed with MPN despite the detection of a JAK2, CALR, or MPL mutation. Among 156 patients with undefined MPN receiving further follow-up, none underwent bone marrow examination and screening for BCR-ABL1 was not performed in 88 cases. Thus, diagnosis was not possible in these cases due to a lack of essential examinations. Since the prognosis and treatment strategy associated with MPN differ among disease types, in addition to mutation analysis, the importance of bone marrow examination and screening for BCR-ABL1 must be re-recognized.

Classical Philadelphia-negative myeloproliferative neoplasms include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). They are characterized by the presence of driver mutations of JAK2, CALR or MPL genes. Overexpression of WT1 is used as a marker of minimal residual disease in acute myeloid leukemia, especially after allogeneic stem cell transplantation (SCT). We investigated WT1 expression at diagnosis in 152 MPN patients and showed that the WT1 transcript was overexpressed in PMFs and PVs compared to controls. In particular, WT1 transcript levels were higher in PMF than in ET and PV. WT1 transcript levels were significantly increased during myelofibrotic transformation of ET or PV. Using multivariate linear regression, high WT1 transcript levels in PMF were associated with age over 65, splenomegaly and thrombocytopenia. The ROC curve analysis showed that a level of WT1 transcript >10 WT1 copies/10⁴ABL1 enabled the diagnosis of PMF with a specificity of 95.8% (PMF vs ET; ROC AUC = 0.91). In myelofibrosis, studying follow-ups of WT1 transcript showed that this marker is of interest after allogeneic SCT. These results demonstrate that WT1 overexpression is a simple marker of myelofibrosis in MPN and could be used during patient follow-up.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell (HSC) disorders that can be classified on the basis of genetic, clinical, phenotypic features. Genetic lesions such as JAK2 mutations and BCR-ABL translocation are often mutually exclusive in MPN patients and lead to essential thrombocythemia, polycythemia vera, or myelofibrosis or chronic myeloid leukemia, respectively. Nevertheless, coexistence of these genetic aberrations in the same patient has been reported. Whether these aberrations occur in the same stem cell or a different cell is unclear, but an unstable genome in the HSCs seems to be the common antecedent. In an effort to characterize the underlying genetic events that might contribute to the appearance of more than one MPN in a patient, we studied neoplastic cells from patients with dual MPNs by next-generation sequencing. We observed that most patients with two MPNs harbored mutations in genes known to contribute to clonal hematopoiesis through altered epigenetic regulation such as TET2, ASXL1/2, SRSF2, and IDH2 at varying frequencies (1%-47%). In addition, we found that some patients also harbored oncogenic mutations in N/KRAS, TP53, BRAF, EZH2, and GNAS at low frequencies, which probably represent clonal evolution. These findings support the hypothesis that hematopoietic cells from MPN patients harbor multiple genetic aberrations, some of which can contribute to clonal dominance. Acquiring mutations in JAK2/CALR/MPL or the BCR-ABL translocation probably drive the oncogenic phenotype towards a specific MPN. Further, we propose that the acquisition of BCR-ABL in these patients is frequently a secondary event resulting from an unstable genome.

Primary myelofibrosis (PMF) is a rare chronic BCR-ABL1-negative myeloproliferative neoplasm characterized by progressive bone marrow fibrosis, inefficient hematopoiesis, and shortened survival. The clinical manifestations of PMF include splenomegaly, consequent to extramedullary hematopoiesis, pancytopenias, and an array of potentially debilitating constitutional symptoms. The diagnosis is based on bone marrow morphology and clinical criteria. Mutations in the JAK2 (V617F), MPL (W515), and CALR (exon 9 indel) genes are found in approximately 90% of patients whereas the remaining 10% are so-called triple negatives. Activation of the JAK/STAT pathway results in overproduction of abnormal megakaryocytes leading to bone marrow fibrosis. These mutations might be accompanied by other mutations, such as ASXL1. The commonly used prognostication scoring for PMF is based on the International Prognostic Scoring System. The subsequently developed Dynamic International Prognostic Scoring System-plus employs clinical as well as cytogenetic variables. In PMF, CALR mutation is associated with superior survival and ASXL1 with inferior outcome. Patients with triple-negative PMF have a higher incidence of leukemic transformation and lower overall survival compared with CALR- or JAK2-mutant patients. The impact of genetic lesions on survival is independent of current prognostic scoring systems. These observations indicate that driver and passenger mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials.

Myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoiesis characterized by a high frequency of genetic alterations, and include chronic myeloid leukemia (CML) and the BCR-ABL1-negative MPNs. Herein we summarize recent advances and controversies in our understanding of the biology and therapy of these disorders, as discussed at the 8th post-American Society of Hematology CML-MPN workshop. The principal areas addressed include the breakthrough discovery of CALR mutations in patients with JAK2/MPL wild type MPN, candidate therapies based on novel genetic findings in leukemic transformation and new therapeutic targets in MPNs, and an appraisal of bone marrow histopathology in MPNs with a focus on the potential new clinical entity of "masked" polycythemia vera. An update on clinical trials of Janus kinase (JAK) inhibitors is presented as well as current understanding regarding the definitions and mechanisms of resistance to JAK inhibitors, and updated information on the safety and efficacy of discontinuation of tyrosine kinase inhibitors in patients with CML.

Suspicion of myeloproliferative neoplasms (MPNs) and especially essential thrombocythemia (ET) in primary care is often based solely on blood counts, with patients referred to a haematologist without a thorough evaluation. We retrospectively assessed the role of calreticulin gene (CALR) mutations in the diagnosis of MPN in this population. We studied CALR mutations in 524 JAK2 V617F-negative patients with suspected MPN. Uncommon CALR mutations were confirmed by Sanger sequencing and searched for in the COSMIC or HGMD database. Mutations were defined as frameshift or non-frameshift mutations. CALR mutations were detected in 23 patients (23/524 = 4.4%). Four mutations detected in our study were newly identified mutations. Non-frameshift mutations were detected in two patients. Most patients (380/524 = 72.5%) were diagnosed with secondary conditions leading to blood count abnormalities such as iron deficiency, inflammatory and infectious diseases, malignancy and hyposplenism. Nine patients (9/23 = 39%) were retrospectively diagnosed with ET based on CALR mutation confirmation. Two patients with non-frameshift CALR mutations were diagnosed with reactive thrombocytosis and MPN unclassifiable, respectively. Our study showed that CALR mutations are important, non-invasive diagnostic indicators of ET and can aid in its diagnosis. Moreover, the type of CALR mutation must be accurately defined, as non-frameshift mutations may not be associated with ET. Finally, CALR mutation detection should be reserved for patients with high suspicion of clonal haematological disease.

BACKGROUND

Mutations in calreticulin (CALR) have been reported to be key markers in the molecular diagnosis of myeloid proliferative neoplasms. In most previous reports, CALR mutations were analyzed by using Sanger sequencing. Here, we report a new, rapid, and convenient system for screening CALR mutations without sequencing.

METHODS

Eighty-three bone marrow samples were obtained from 81 patients with thrombocytosis. PCR primers were designed to detect wild-type CALR (product: 357 bp) and CALR with type 1 (product: 302 bp) and type 2 mutations (product: 272 bp) in one reaction. The results were confirmed by Sanger sequencing and compared with results from fragment analysis.

RESULTS

The minimum detection limit of the screening PCR was 10 ng for type 1, 1 ng for type 2, and 0.1 ng for cases with both mutations. CALR type 1 and type 2 mutants were detected with screening PCR with a maximal analytical sensitivity of 3.2% and <0.8%, respectively. The screening PCR detected 94.1% (16/17) of mutation cases and showed concordant results with sequencing in the cases of type 1 and type 2 mutations. Sanger sequencing identified one novel mutation (c.1123_1132delinsTGC). Compared with sequencing, the screening PCR showed 94.1% sensitivity, 100.0% specificity, 100.0% positive predictive value, and 98.5% negative predictive value. Compared with fragment analysis, the screening PCR presented 88.9% sensitivity and 100.0% specificity.

CONCLUSIONS

This screening PCR is a rapid, sensitive, and cost-effective method for the detection of major CALR mutations.

Alterations in the bone marrow niche induced by abnormal production of cytokines and other soluble factors have been associated with disease progression in classical BCR-ABL1 negative myeloproliferative neoplasms (MPN). Variations in circulating proteins might reflect local disease processes and plasma proteome profiling could serve to identify possible diagnostic and prognostic biomarkers. We employed a human cytokine array to screen for 105 distinct analytes in pooled plasma samples obtained from untreated young MPN patients (<35 years) with different clinical phenotypes and driver mutations, as well as from healthy individuals. Among molecules that exhibited significantly increased levels in MPN patients versus controls, the top of the list was represented by Dickkopf-related protein 1 (Dkk-1), which also showed the highest potential for discrimination between MPN subtypes. In the next step, a quantitative ELISA was used to measure plasma Dkk-1 levels in 30 young-onset MPN-10 essential thrombocythemia (ET), 10 polycythemia vera (PV), 10 pre-fibrotic primary myelofibrosis (pre-PMF)-and 10 controls. The results suggested that plasma Dkk-1 levels could differentiate ET from pre-PMF, in JAK2 V617F-positive as well as in CALR-positive patients, and also ET from PV in JAK2 V617F-positive patients.

Different intrinsic and extrinsic stress pathways including endoplasmic reticulum (ER) stress converge on the phosphorylation of eukaryotic translation initiation factor 2A (EIF2A, best known as eIF2α), which characterizes the so-called "integrated stress response". This phosphorylation event is important for the induction of autophagy in response to multiple distinct stressors, as well as for the exposure of calreticulin (CALR) as an "eat me" signal on the surface of the plasma membrane of stressed cells. Both autophagy and CALR exposure are required for immunogenic cell death, a modality of cellular demise that ignites anticancer and antiviral immune responses. In several different cancer types, eIF2α phosphorylation indicates favorable prognosis, correlating with an enhanced antitumor immune response.

Keywords: Integrated stress response; antitumor immune response; autophagosome; calreticulin; endoplasmic reticulum stress.

Immunogenic cell death (ICD), induced by certain anticancer chemotherapeutics, leads to the emission of danger associated molecular patterns (DAMP) by cancer cells, which facilitates the attraction, activation and maturation of dendritic cells (DC) as well as the subsequent priming of effector T cells. In this context calreticulin (CALR) exposed at an early stage of ICD at the surface of the cancer cells serves as phagocytic signal and triggers the formation of immunological synapses between malignant cells and DC. Subsequent phagocytosis facilitates the transfer of tumor associated antigen and thus depicts a fundamental step in the generation of anticancer immunity. Here we provide an imaging flowcytometric protocol for the quantification of ICD-associated DC phagocytosis of cancer cells. As compared to the traditional flowcytometry-based analysis, the presented method offers additional means of differentiation between the transient formation of immunological synapses and the final DC-mediated phagocytosis of cancer cells.

The genetic events associated with transformation of myeloproliferative neoplasms (MPNs) to secondary acute myeloid leukemia (sAML), particularly in the subgroup of essential thrombocythemia (ET) patients, remain incompletely understood. Deep studies using high-throughput methods might lead to a better understanding of genetic landscape of ET patients who transformed to sAML. We performed array-based comparative genomic hybridization (aCGH) and whole exome sequencing (WES) to analyze paired samples from ET and sAML phases. We investigated five patients with previous history of MPN, which four had initial diagnosis of ET (one case harboring JAK2 p.Val617Phe and the remaining three CALR type II p.Lys385fs*47), and one was diagnosed with MPN/myelodysplastic syndrome with thrombocytosis (SF3B1 p.Lys700Glu). All were homogeneously treated with hydroxyurea, but subsequently transformed to sAML (mean time of 6 years/median of 4 years to transformation). Two of them have chromosomal abnormalities, and both acquire 2p gain and 5q deletion at sAML stage. The molecular mechanisms associated with leukemic progression in MPN patients are not clear. Our WES data showed TP53 alterations recurrently observed as mutations (missense and frameshift) and monoallelic loss. On the other hand, aCGH showed novel chromosome abnormalities (+2p and del5q) potentially associated with disease progression. The results reported here add valuable information to the still fragmented molecular basis of ET to sAML evolution. Further studies are necessary to identify minimal deleted/amplified region and genes relevant to sAML transformation.

Ovarian cancer is prevalent in women which is usually diagnosed at an advanced stage with a high mortality rate. The aim of this study is to investigate protein-coding gene, long non-coding RNA, and microRNA associated with the prognosis of patients with ovarian serous carcinoma by mining data from TCGA (The Cancer Genome Atlas) public database. The clinical data of ovarian serous carcinoma patients was downloaded from TCGA database in September, 2016. The mean age and survival time of 407 patients with ovarian serous carcinoma were 59.71 ± 11.54 years and 32.98 ± 26.66 months. Cox's proportional hazards regression analysis was conducted to analyze genes that were significantly associated with the survival of ovarian serous carcinoma patients in the training group. Using the random survival forest algorithm, Kaplan-Meier and ROC analysis, we kept prognostic genes to construct the multi-dimensional transcriptome signature with max area under ROC curve (AUC) (0.69 in the training group and 0.62 in the test group). The selected signature composed by VAT1L, CALR, LINC01456, RP11-484L8.1, MIR196A1 and MIR148A, separated the training group patients into high-risk or low-risk subgroup with significantly different survival time (median survival: 35.3 months vs. 64.9 months, P < 0.001). The signature was validated in the test group showing similar prognostic values (median survival: 41.6 months in high-risk vs. 57.4 months in low-risk group, P=0.018). Chi-square test and multivariable Cox regression analysis showed that the signature was an independent prognostic factor for patients with ovarian serous carcinoma. Finally, we validated the expression of the genes experimentally.

Mutations in exon 9 of the calreticulin gene (CALR) frequently occur in patients with chronic myeloproliferative neoplasms (MPN). Patients exhibit spontaneous cellular immune responses to epitopes derived from the mutant CALR C-terminus, and CALR-mutant-specific T cells recognize autologous CALR-mutant malignant cells. This study investigated whether CALR-mutant-specific T cells occur naturally in CALRwt MPN-patients and in healthy individuals. Specific immune responses against epitopes in the mutant CALR peptide sequence were detected in both CALRwt MPN-patients and in healthy individuals. Healthy donors displayed more frequent and stronger CALR-mutant specific T-cell responses compared to the responses identified in CALR-mutant MPN-patients. Several T-cell responses were identified in healthy donors directly ex vivo. Importantly, by running functional analyses on live-sorted immune cells from healthy donors, we showed that circulating CALR-mutant-specific immune cells are T-memory cells. These findings suggest, that healthy individuals acquire a CALR exon 9 mutation, but the immune system reacts and clears the mutant cells, and during this reaction generates CALR-mutant specific T-memory cells. We believe that these findings provide the evidence for tumor immune surveillance in MPN.

UNASSIGNED

The main goal of this analysis was prioritization of co-expressed genes and miRNAs that are thought to have important influences in the pathogenesis of colon and lung cancers.

UNASSIGNED

MicroRNAs (miRNAs) as small and endogenous noncoding RNAs which regulate gene expression by repressing mRNA translation or decreasing stability of mRNAs; they have proven pivotal roles in different types of cancers. Accumulating evidence indicates the role of miRNAs in a wide range of biological processes from oncogenesis and tumor suppressors to contribution to tumor progression. Colon and lung cancers are frequently encountered challenging types of cancers; therefore, exploring trade-off among underlying biological units such as miRNA with mRNAs will probably lead to identification of promising biomarkers involved in these malignancies.

UNASSIGNED

Colon cancer and lung cancer expression data were downloaded from Firehose and TCGA databases and varied genes extracted by DCGL software were subjected to build two gene regulatory networks by parmigene R package. Afterwards, a network-driven integrative analysis was performed to explore prognosticates genes, miRNAs and underlying pathways.

UNASSIGNED

A total of 192 differentially expressed miRNAs and their target genes within gene regulatory networks were derived by ARACNE algorithm. BTF3, TP53, MYC, CALR, NEM2, miR-29b-3p and miR-145 were identified as bottleneck nodes and enriched via biological gene ontology (GO) terms and pathways chiefly in biosynthesis and signaling pathways by further screening.

UNASSIGNED

Our study uncovered correlated alterations in gene expression that may relate with colon and lung cancers and highlighted the potent common biomarker candidates for the two diseases.

The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), which include essential thrombocythemia, polycythemia vera, and myelofibrosis (MF), are in a new era of molecular diagnosis, ushered in by the identification of the JAK2(V617F) and cMPL mutations in 2005 and 2006, respectively, and the CALR mutations in 2013. Coupled with increased knowledge of disease pathogenesis and refined diagnostic criteria and prognostic scoring systems, a more nuanced appreciation has emerged of the burden of MPN in the United States, including the prevalence, symptom burden, and impact on quality of life. Biological advances in MPN have translated into the rapid development of novel therapeutics, culminating in the approval of the first treatment for MF, the JAK1/JAK2 inhibitor ruxolitinib. However, certain practical aspects of care, such as those regarding diagnosis, prevention of vascular events, choice of cytoreductive agent, and planning for therapies, present challenges for hematologists/oncologists, and are discussed in this article.

OBJECTIVE

Adverse early-life experiences have been suggested as one of the key contributors to neurodevelopmental disorders, such that these experiences influence brain development, cognitive ability and mental health. Previous studies indicated that hippocampal levels of the calcium-binding proteins calretinin (CALR) and calbindin-D28k (CALB) changed in response to maternal deprivation (MD), a model for adverse early-life experiences. We investigated the effects of MD on hippocampal CALR and CALB protein levels and cognitive behaviors, and explored whether these effects were sex-related.

METHODS

From postnatal day 2 (PND-2) to PND-14, rat pups in the MD group were separated from their mothers for 3 h/day for comparison with pups raised normally (control). To determine hippocampal CALR and CALB levels, fluorescent immunostaining of hippocampal sections and Western blot analysis of hippocampal tissues were employed at various timepoints (PND-21, -25, -30, -35 and -40). Behavioral and cognitive changes were determined by open field test (PND-21) and Morris water maze (PND-25).

RESULTS

Western blot analysis showed changes in the hippocampal CALR and CALB levels in both male and female MD groups, compared with controls. The open field test showed reduced exploration only in male MD groups but not female MD groups. The Morris water maze tests indicated that MD caused spatial memory impairment both in male and female rats, but there was a sex difference in CALR and CALB levels.

CONCLUSIONS

Male rats are relatively more vulnerable to MD stress than female rats, but both male and female rats demonstrate spatial learning impairment after exposure to MD stress. Sex difference in CALR and CALB levels may reveal the different mechanisms behind the behavioral observations.

Activation of the phagocytosis of macrophages to tumor cells is an attractive strategy for cancer immunotherapy, but the effectiveness is limited by the fact that many tumor cells express an increased level of anti-phagocytic signals (e.g., CD47 molecules) on their surface. To promote phagocytosis of macrophages, a pro-phagocytic nanoparticle (SNPA_CALR&aCD47 ) that concurrently carries CD47 antibody (aCD47) and a pro-phagocytic molecule calreticulin (CALR) is constructed to simultaneously modulate the phagocytic signals of macrophages. SNPA_CALR&aCD47 can achieve targeted delivery to tumor cells by specifically binding to the cell-surface CD47 and block the CD47-SIRPα pathway to inhibit the "don't eat me" signal. Tumor cell-targeted delivery increases the exposure of recombinant CALR on the cell surface and stimulates an "eat me" signal. Simultaneous modulation of the two signals enhances the phagocytosis of 4T1 tumor cells by macrophages, which leads to significantly improved anti-tumor efficacy in vivo. The findings demonstrate that the concurrent blockade of anti-phagocytic signals and activation of pro-phagocytic signals can be effective in macrophage-mediated cancer immunotherapy.

Keywords: CD47-SIRPα pathway; cancer immunotherapy; macrophages; pro-phagocytic nanoparticles.

BACKGROUND

The discovery of somatic mutations within the gene encoding calreticulin (CALR) in 2013 represented a major milestone in the molecular diagnosis of BCR-ABL negative myeloproliferative neoplasms (MPN). In fact, exome sequencing revealed that most patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) lacking JAK2 or MPL mutations, harbor somatic insertion and/or deletion in exon 9 of CALR gene. In this study, we identified the first CALR gene mutational landscape in Moroccan patients with MPN nonmutated for the JAK2 gene.

METHODS

We performed Sanger sequencing of exon 9 of CALR gene in blood samples obtained from 33 Moroccan patients with ET or PMF non-mutated for JAK2.

RESULTS

Of the 33 patients analyzed, we detected eight distinct variants in 15 patients (45.4%); six indel mutations, five with type 1 recurrent 52bp deletion, four with type 2 recurrent 5bp insertion and one in frame deletion which was found to be a germline variant suggesting a very rare condition in MPN.

CONCLUSIONS

This is the first cohort reported in CALR gene mutation analysis in Morocco. Our results were concordant with studies reported up to date and very encouraging in promoting the molecular diagnosis of myeloproliferative neoplasms in Moroccan patients. Moreover, the presence of a germline in frame deletion in a symptomatic patient should undermine the effectiveness of sizing assays without DNA sequencing in the diagnosis of CALR mutations.

Somatic mutations of Janus kinase 2 (JAK2V617F), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) are the major clonal molecules that drive the pathogenesis of myeloproliferative neoplasms (MPN). It is well recognized that MPN patients carry an excessive risk of thrombohemorrhagic complications. However, little is known about the prevalence of these clonal markers in patients with cerebral vascular disease.

To address this issue, 153 consecutive stroke patients in Taiwan were enrolled in the study. Allele-specific PCR (AS-PCR), real-time AS-PCR, and Illumina paired-end sequencing were employed to detect the presence of MPL, JAK2V617F, and CALR exon 9 mutations, respectively.

JAK2V617F mutation was detectable in 13 samples (8.5%), but the allele burdens (AB) were greater than 1% in only six (3.9%) of them. Compared to JAK2-unmutated patients, those with JAK2V617F AB > 1% had significantly higher white blood count (p = 0.01), although four of the six did not exhibit MPN phenotypes. Two patients had a heterozygous CALR exon9 mutation locating outside the coding region and did not alter the amino acid sequence of this protein. On the other hand, there were no patients carrying the MPL mutations. Using patient age, baseline hemogram, and stroke-relevant risk factors, we developed a predictive model that could successfully identify stroke patients at risk of carrying clonal JAK2V617F mutation.

The prevalence of JAK2V617F mutation in stroke patients was higher than that seen in general population. Based on our newly developed probability stratification model, genotyping of JAK2V617F mutation in selected patients with stroke might be warranted.

Peripheral blood sample was donated by a 61years old female patient diagnosed with acute myeloid leukemia secondary to a primary myelofibrosis harboring the 52-bp deletion in the CALR gene (c.1092_1143del, p.L367fs*46) and the R693X mutation in the ASXL1 gene (c.2077C>T, p.R693X). CD34+ cells were isolated from the sample and subjected to the reprogramming procedure by using the Sendai virus carrying the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc. iPS colonies generated retained the original mutations and displayed all the features of bona fide iPS cells.

Somatic mutations in the calreticulin (CALR) gene have been found in most patients with JAK2- and MPL-unmutated Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). It has recently been shown that mutant CALR constitutively activates the thrombopoietin receptor MPL and, thus, plays a causal role in the development of MPNs. However, the roles of mutant CALR in human haematopoietic cell differentiation remain predominantly elusive. To examine the impact of the 5-base insertion mutant CALR gene (Ins5) on haematopoietic cell differentiation, we generated induced pluripotent stem cells from an essential thrombocythaemia (ET) patient harbouring a CALR-Ins5 mutation and from a healthy individual (WT). Megakaryopoiesis was more prominent in Ins5-haematopoietic progenitor cells (Ins5-HPCs) than in WT-HPCs, implying that the system recapitulates megakaryocytosis observed in the bone marrow of CALR-mutant ET patients. Ins5-HPCs exhibited elevated expression levels of GATA1 and GATA2, suggesting a premature commitment to megakaryocytic differentiation in progenitor cells. We also demonstrated that 3-hydroxy anagrelide markedly perturbed megakaryopoiesis, but not erythropoiesis. Collectively, we established an in vitro model system that recapitulates megakaryopoiesis caused by mutant CALR. This system can be used to validate therapeutic compounds for MPN patients harbouring CALR mutations and in detailed studies on mutant CALR in human haematological cell differentiation.