scholarly journals Modeling Calreticulin-Mutant Myeloproliferative Neoplasms with Isogenic Induced Pluripotent Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4319-4319 ◽  
Author(s):  
Wei Wang ◽  
Tiansu Wang ◽  
Andriana G. Kotini ◽  
Camelia Iancu-Rubin ◽  
Ronald Hoffman ◽  
...  
Keyword(s):  
Research Funding ◽  
Ex Vivo ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Mutant Type ◽  
C Terminus ◽  
Calr Mutations

Abstract Myeloproliferative neoplasms (MPN) are characterized by the excessive production of one or more myeloid lineages and a propensity to progress to acute leukemia. In 2013, mutations in the CALR gene, encoding calreticulin, were identified in patients with MPN, mutually exclusive to the previously identified JAK2 and MPL (TPO-R) mutations. CALR mutations are frameshift mutations - typically a 52-bp deletion (type 1) or a 5-bp insertion (type 2) - that result in a novel C-terminus. The discovery of mutations in a ubiquitously expressed multifunctional protein like calreticulin was unanticipated. Subsequent studies found that CALR mutations lead to activation of JAK/STAT, mediated through aberrant interactions between mutant CALR and MPL, thus presenting an excellent opportunity for targeted therapy. However, the mechanism of MPL activation remains largely unexplained with prior studies using cell lines with exogenous expression of CALR and MPL following transfection. To create a more physiological cellular model to study the effects of CALR mutations, we established multiple iPSC lines from two patients with CALR-mutant MPN - one type 1-like (del34) and one type 2 (ins5) -, as well as from one patient with JAK2V617F MPN. All iPSC lines were confirmed to harbour the CALR or JAK2V617F mutation found in the corresponding patient, to express mutant calreticulin, as detected by flow cytometry using an antibody which specifically recognizes the novel calreticulin C-terminus, and to be karyotypically normal. Genetically matched iPSC lines with WT JAK2 could also be generated from the JAK2V617F (but not the CALR-mutant) patient cells in the same reprogramming round. CRISPR gene editing was used to generate isogenic CALR-corrected lines from both CALR-mutant patients. Furthermore, in order to facilitate biochemical studies, we used CRISPR to introduce a V5 epitope tag in one allele of the endogenous mutant or WT CALR gene, in mutant and isogenic corrected iPSC lines, respectively. We optimized an in vitro differentiation protocol for efficient derivation of megakaryocyte (MK) progenitors from iPSCs and found disease-relevant phenotypes, mainly TPO-independent MK colony formation in semi-solid media, which is the phenotypic hallmark of ex vivo primary MPN cells. In the absence of TPO, JAK2 V617F, CALR-mutant type 1-like and CALR-mutant type 2 iPSCs generated 52.1%, 58.7±22.2% and 59.8±3.6%, respectively, of the number of MK colonies generated in the presence of TPO, as opposed to 10%, 8.8±1.8% and 0.5±0.9%, respectively, for the matched WT JAK2, the corrected CALR-mutant type 1-like and the corrected CALR-mutant type 2 iPSCs. Isolated CALR mutant iPSC-derived CD41a+ MK progenitors had increased phosphorylation of STAT5 following cytokine starvation as compared to isogenic corrected and non-isogenic normal cells. CALR-mutant cells expressed equal transcript levels of the WT and mutant CALR alleles. However, mutant CALR protein levels were severely reduced, at levels 1~12% of those of the WT protein. This is consistent with previous studies documenting instability of mutant calreticulin. Transcriptomics (RNA-seq) and proteomics analyses of CD41a+-sorted MK progenitors derived from CALR mutant and isogenic corrected iPSCs are ongoing. These iPSC models offer the opportunity to study the effects of CALR mutations in a cellular context with both MPL and CALR (WT or mutant) expressed from their endogenous loci. They thus provide a powerful platform to investigate the disease mechanisms underlying CALR-mutant MPNs and to perform small molecule and genetic (CRISPR) screens to identify new therapeutic targets. Disclosures Iancu-Rubin: Merck: Research Funding; Incyte: Research Funding; Summer Road, LLC: Research Funding; Formation Biologics: Research Funding. Hoffman:Incyte: Research Funding; Merus: Research Funding; Formation Biologics: Research Funding; Janssen: Research Funding; Summer Road: Research Funding.

Mutation Type As a Major Determinant of Clinical Phenotype in Myeloproliferative Neoplasms Associated with Mutant Calreticulin

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3215-3215 ◽  
Author(s):  
Daniela Pietra ◽  
Elisa Rumi ◽  
Chiara Milanesi ◽  
Christian A Di Buduo ◽  
Marta Bellini ◽  
...  
Keyword(s):  
Amino Acids ◽  
Clinical Phenotype ◽  
Myeloproliferative Neoplasms ◽  
Jak2 V617f ◽  
Charged Amino Acids ◽  
Calr Mutations ◽  
Mutation Type ◽  
Calr Mutation

Abstract About 25% of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) carry a somatic mutation of CALR, the calreticulin gene [N Engl J Med. 2013;369:2379-90]. So far, more than 50 different indels in CALR exon 9 have been found, but a 52-bp deletion (type 1 mutation) and a 5-bp insertion (type 2) are the most common lesions. All indels generate a novel C-terminus of the mutant protein, in which the endoplasmic reticulum retention signal KDEL is lost and the negatively charged amino acids are replaced by neutral and positively charged amino acids, disrupting the Ca-binding site. This suggests that both cellular dislocation and impaired Ca-binding activity may be involved in the abnormal proliferation of cells expressing a mutant calreticulin. It is still unclear, however, why the same mutant gene is associated with 2 different disease phenotypes (ET and PMF). In particular, little in known about the effect of the mutant protein on megakaryocyte biology and bone marrow collagen deposition. We studied the relationships between CALR mutation type, megakaryocyte biology, and clinical phenotype in patients with myeloproliferative neoplasms. According to the 2008 WHO criteria, 716 out of 892 patients had ET and 176 had PMF. Overall, 578 (65%) patients carried JAK2 (V617F), 230 (26%) had a CALR indel, and 84 (9%) had nonmutated JAK2 and CALR. Patients with MPL mutations were excluded. Twenty-six different types of CALR lesions were identified: 120 (52%) patients had type 1 mutation, 75 (33%) had type 2, and 35 (15%) carried other indels. The frequency of type 1 mutation was significantly higher in PMF than in ET (71% vs 46%, P=.004). All these variants involved 3 different stretches of negatively charged amino acids, with an increase in the isoelectric points (pI) of the mutant protein. As type 1 and type 2 mutations affected stretch I and III, respectively, the 26 indels were categorized into 3 groups on the basis of the stretch they affected: i) type 1-like (61%), affecting stretch I; ii) type 2-like (36%), stretch III; iii) and other types (3%), stretch II. The pI values were significantly different in the 3 groups (P<.001). The frequency of type-1 like mutations was significantly higher in PMF than in ET (82% vs 55%, P=.001). In vitro differentiated megakaryocytes from CALR-mutant patients displayed a significant increase in the extent of both intracellular Ca2+ release from the endoplasmic reticulum and extracellular Ca2+ entry inside the cytoplasm, as compared with healthy controls. Megakaryocytes carrying type 1-like CALR mutations exhibited the highest amplitude of Ca2+ flows regardless of the type of disease. In ET, impaired Ca2+ homeostasis was accompanied by atypical proplatelet architecture (ie, more branches and bifurcations). With respect to clinical phenotype at diagnosis, ET patients with type 2-like CALR mutation showed a trend towards higher PLT count (P=.063) and lower age (P=.053), and significantly lower LDH values (P=.021) than those with type 1-like mutation. In a hierarchical cluster analysis including demographic, clinical and molecular data, CALR mutation type (1 vs 2) identified the 2 clusters with the highest dissimilarity. Considering all patients, those with type 2-like CALR lesions had a better survival than those with JAK2 (V617F) (96.1% vs 84.4% at 10 years, P=.039), while no difference was found between the 2 CALR mutation types. ET patients with type 2-like CALR mutations showed a lower risk of thrombosis than those with JAK2 (V617F) (P=.010). By contrast, ET patients with type 1-like CALR mutations had a higher risk of myelofibrotic transformation that those with type 2-like CALR mutations (P=.029) and especially those with JAK2 (V617F) (P=.011). Finally, PMF patients with type 1-like CALR variants had a better survival than those with JAK2 (V617F) (80.1% vs 48% at 10 years, P=.008). In summary, abnormalities in megakaryocyte calcium metabolism and proplatelet architecture are found in patients with CALR-mutant myeloproliferative neoplasms, and their extent is related to mutation type. Type 2-like CALR mutations are more likely to be associated with isolated thrombocytosis without bone marrow fibrosis, ie, with an ET phenotype. By contrast, type 1-like CALR mutations are generally associated with bone marrow fibrosis, ie, with a PMF phenotype. Thus, in CALR-mutant myeloproliferative neoplasms, the mutation type is a major determinant of the clinical phenotype. Disclosures No relevant conflicts of interest to declare.

scholarly journals Calreticulin mutations Are Present in Polyclonal As Well As Clonal ET

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4590-4590
Author(s):  
Xylina Gregg ◽  
Sabina Swierczek ◽  
Soo Jin Kim ◽  
Josef T. Prchal
Keyword(s):  
T Cells ◽  
X Chromosome ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Hematopoietic Stem ◽  
Clonal Hematopoiesis ◽  
Calr Mutations ◽  
Calr Mutation

Abstract First and second authors contributed equally During female embryogenesis, most of the genes in either the maternal or paternal X-chromosome are randomly inactivated in each cell, a process that remains remarkably constant in their progeny. X-chromosome inactivation has been used to define clonality in myeloproliferative neoplasms (MPNs) such polycythemia vera (PV), primary myelofibrosis (PMF) and essential thrombocythemia (ET). One such method to determine clonality uses a quantitative, transcriptional clonality assay based on conservative exonic polymorphisms in five X-chromosome genes (MPP1, FHL1, IDS, BTK, and G6PD). Females who are heterozygous for any of these polymorphisms are considered “informative” and can be studied for clonality by interrogating their platelets’ and granulocytes’ RNA allelic usage ratio. JAK2 mutations occur in >95% of PV and 50-60% of ET and PMF; cMPL mutations are found in another 5-10% of ET and MF. Somatic calreticulin (CALR) mutations have been identified in a majority of patients with ET and MF who lack JAK2 and cMPL mutations. CALR mutations are reported to be associated with a more favorable prognosis and are believed to be acquired early in the disease course. More than 30 CALR mutations have been described, but type 1 (52-bp deletion; c.1092_1143del) and type 2 (5-bp insertion; c.1154_1155insTTGTC) mutations are the most frequent. We analyzed 61 females informative for a transcriptional clonality assay and 44 males with unexplained thrombocytosis or marrow fibrosis and no detectable JAK2 or cMPL mutations for CALR mutations in their granulocytes. With the exception of an absence of a clonal marker, these patients met WHO criteria for ET or PMF. A CALR mutation (20 type 1 and 17 type 2) was present in 37 of these 105 patients (22 females and 15 males). One of the CALR mutated females had a paternal grandmother with JAK2V617F –positive PV, confirming a previous report that, in familial clustering of MPNs, affected individuals may carry different disease-defining somatic mutations. In those CALR positive patients who had available T cells, no detectable CALR mutations were found in their T cells. In one of these subjects, CD34+ cells were available and had a similar mutation level as in the granulocytes. Of the 22 females with a CALR mutation, 19 had clonal hematopoiesis, but 3 had polyclonal hematopoiesis; all 3 had previously unexplained thrombocytosis. None of these patients had any prior treatment for thrombocytosis. Clonal hematopoiesis was present in 26 of the 39 females without a CALR mutation. All female patients with myelofibrosis had clonal hematopoiesis, regardless of CALR mutation status. In contrast to the polyclonal hematopoiesis seen in some CALR positive ET patients, 166 informative PV and JAK2V617F-positive ET or PMF females all had clonal hematopoiesis. We report that CALR mutations are associated with polyclonal hematopoiesis in some ET patients. This finding differs from JAK2V617F-positive ET and PMF and PV females, where clonal hematopoiesis was always seen. This indicates that CALR mutated clones have a weaker suppressive effect on residual normal hematopoietic stem cells than JAK2 mutated clones and may contribute to the possibly more benign course of CALR mutated ET. The CALR mutation was not detected in T cells, which also differs from JAK2V617F mutated MPNs, where a small level of the JAK2 mutation is often detected in T cells. Similar to other reports, we found a lower prevalence of the CALR mutation in JAK2 or cMPL non-mutated ET and PMF than initially described. Disclosures No relevant conflicts of interest to declare.

Comparative expression of hCG β-genes in human trophoblast from early and late first-trimester placentas

2012 ◽  
Vol 303 (8) ◽  
pp. E950-E958 ◽  
Author(s):  
M. Cocquebert ◽  
S. Berndt ◽  
N. Segond ◽  
J. Guibourdenche ◽  
P. Murthi ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Primary Cultures ◽  
First Trimester ◽  
Maternal Serum ◽  
Human Trophoblast ◽  
Intervillous Space ◽  
Β Hcg

Human chorionic gonadotropin (hCG) displays a major role in pregnancy initiation and progression and is involved in trophoblast differentiation and fusion. However, the site and the type of dimeric hCG production during the first trimester of pregnancy is poorly known. At that time, trophoblastic plugs present in the uterine arteries disappear, allowing unrestricted flow of maternal blood to the intervillous space. The consequence is an important modification of the trophoblast environment, including a rise of oxygen levels from about 2.5% before 10 wk of amenorrhea (WA) to ∼8% after 12 WA. Two specific β-hCG proteins that differ from three amino acids have been described: type 1 (CGB7) and type 2 (CGB3, -5, and -8). Here, we demonstrated in situ and ex vivo on placental villi and in vitro in primary cultures of human cytotrophoblasts that type 1 and 2 β-hCG RNAs and proteins were expressed by trophoblasts and that these expressions were higher before blood enters in the intervillous space (8–9 vs. 12–14 WA). hCG was immunodetected in villous mononucleated cytotrophoblasts (VCT) and syncytiotrophoblast (ST) at 8–9 WA but only in ST at 12–14 WA. Furthermore, hCG secretion was fourfold higher in VCT cultures from 8–9 WA compared with 12–14 WA. Interestingly, VCT from 8–9 WA placentas were found to exhibit more fusion features. Taken together, we showed that type 1 and type 2 β-hCG are highly expressed by VCT in the early first trimester, contributing to the high levels of hCG found in maternal serum at this term.

scholarly journals A Novel Pathogenic CALR Exon 9 Mutation in a Patient with Essential Thrombocythemia

2019 ◽  
Vol 51 (3) ◽  
pp. 306-309
Author(s):  
Jee-Soo Lee ◽  
Ho Young Kim ◽  
Miyoung Kim ◽  
Young Kyung Lee
Keyword(s):  
Essential Thrombocythemia ◽  
Frameshift Mutation ◽  
Myeloproliferative Neoplasms ◽  
Case Reports ◽  
Individual Case ◽  
First Case ◽  
Exon 9 ◽  
Calr Mutations

Abstract The clinical phenotypes and prognoses of CALR-mutant myeloproliferative neoplasms depend on the mutation type. The 2 most common mutations, type 1 (52-bp deletion) and type 2 (5-bp insertion), account for 85% of CALR-mutated neoplasms. The former confers a myelofibrotic phenotype, and the latter is associated with a low risk of thrombosis and an indolent clinical course. Individual case reports for patients with novel pathogenic CALR mutations are rare. Herein, we present the first case in the literature, to our knowledge, of a 63-year old ethnic Korean man with essential thrombocythemia who was diagnosed with a novel +1-bp frameshift mutation in CALR, which was predicted to exhibit a type 2–like phenotype.

scholarly journals The Promise of Lung Organoids for Growth and Investigation of Pneumocystis Species

2021 ◽  
Vol 2 ◽  
Author(s):  
Nikeya Tisdale-Macioce ◽  
Jenna Green ◽  
Anne-Karina T. Perl ◽  
Alan Ashbaugh ◽  
Nathan P. Wiederhold ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Culture System ◽  
Ex Vivo ◽  
Alveolar Type ◽  
Proof Of Concept ◽  
3 Dimensional ◽  
Alveolar Epithelial

Pneumocystis species (spp.) are host-obligate fungal parasites that colonize and propagate almost exclusively in the alveolar lumen within the lungs of mammals where they can cause a lethal pneumonia. The emergence of this pneumonia in non-HIV infected persons caused by Pneumocystis jirovecii (PjP), illustrates the continued importance of and the need to understand its associated pathologies and to develop new therapies and preventative strategies. In the proposed life cycle, Pneumocystis spp. attach to alveolar type 1 epithelial cells (AEC1) and prevent gas exchange. This process among other mechanisms of Pneumocystis spp. pathogenesis is challenging to observe in real time due to the absence of a continuous ex vivo or in vitro culture system. The study presented here provides a proof-of-concept for the development of murine lung organoids that mimic the lung alveolar sacs expressing alveolar epithelial type 1 cells (AEC1) and alveolar type 2 epithelial cells (AEC2). Use of these 3-dimensional organoids should facilitate studies of a multitude of unanswered questions and serve as an improved means to screen new anti- PjP agents.

scholarly journals The Prognostic Advantage of Calreticulin Mutations in Myelofibrosis Might be Confined to Type 1 or “type 1-like” Calreticulin Variants

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3166-3166
Author(s):  
Ayalew Tefferi ◽  
Terra L Lasho ◽  
Alexander Tischer ◽  
Emnet A Wassie ◽  
Christy Finke ◽  
...  
Keyword(s):  
Leukocyte Count ◽  
Alpha Helix ◽  
Wild Type ◽  
C Terminus ◽  
Helix Propensity ◽  
The Difference ◽  
Calr Mutations ◽  
Propensity Scale

Abstract Background : Approximately 25% of patients with primary myelofibrosis (PMF) harbor calreticulin (CALR) mutations, which have been associated with longer survival (Klampf et al. NEJM 2013). More than 80% of CALR mutated patients harbor one of two mutation variants: type 1, a 52-bp deletion (p.L367fs*46) or type 2, a 5-bp TTGTC insertion (p.K385fs*47). Recent studies have suggested phenotypic and prognostic differences between these two variants (Tefferi et al. Blood 2014, Leukemia 2014 and AJH 2014). Furthermore, data are emerging that suggest functionally-relevant structural differences between type 1 and type 2 CALR variants, including a higher alpha-helix content of the mutant C-terminus in type 2, compared to type 1 (Eder-Azanza et al. Leukemia 2014). Objectives : We used statistical models to calculate helix propensity for thirty-one unique amino acid sequences that were altered by CALR mutations and used the results to subclassify non-type 1/2 CALR mutations into “type 1-like” and “type 2-like” variants. Subsequently, we examined the prognostic relevance of these subgroups. Methods : Calculation of helix propensity, which is the percentage of residues that are predicted to be involved in the formation of an alpha-helix, was performed using AGADIR, which is a statistical approximation algorithm (Munoz et al. Biopolymers 1997). The helix tendency calculations were performed using conditions of pH 7.0, 5 and 25 °C, an ionic strength of 0.1 M and no N- or C-terminal protection. Results : 532 PMF patients were screened for JAK2, CALR and MPL mutations; the respective mutational frequencies were 58%, 24.6% and 7.3%. Among the 131 CALR-mutated cases, 98 (74.8%) harbored type 1, 15 (11.5%) type 2 and 18 (13.7%) other variants. Based on predicted helix propensity scale, the “other” CALR mutations were subclassified as type 1-like (n=12) or type 2-like (n=6) and respectively grouped with type 1 and type 2 variants, for purposes of phenotypic and prognostic comparisons. The AGADIR-derived predicted helix propensity scale was 29.69 for wild-type CALR and 8.6 or 34.17 for type 1 and type 2 mutant CALR, respectively; accordingly, CALR variants with values that are close to or above the value for wild-type CALR were classified as “type 2-like” (range 26.47-36.12) and those with values close to or below the value for type 1 as “type 1-like” (range 2.11-17.3). Comparison of “type 1/type 1-like” (n=110) and “type 2/type 2-like” (n=21) CALR mutations showed the latter to be associated with higher DIPSS-plus score (p=0.01), EZH2 mutations (p<0.01), leukocyte count >25 x 10(9)/L (p<0.01), higher circulating blast percentage (p=0.02) and palpable spleen size >10 cm (p<0.01). Comparison of “type 1/type 1-like” CALR and JAK2 mutations (n=309) showed the former to be associated with younger age, higher platelet count, lower transfusion need, higher hemoglobin level, lower leukocyte count and lower DIPSS-plus score (p<0.01 for all comparisons). None of these associations was evident during comparison of “type 2/type 2-like” CALR with JAK2 mutations. Survival was similar between patients with type 1 and “type 1-like” (p=0.8) and between type 2 and “type 2-like” (p=0.63) CALR mutations. In contrast, survival was significantly shorter in patients with type 2 (HR 2.4, 95% CI 1.2-4.8) and “type 2-like” (HR 3.2, 95% CI 1.0-10.6), when compared to those with type 1 CALR mutations. Survival was also significantly shorter with “type 2/type 2-like” vs “type 1/type 1-like” CALR mutations (p=0.003; HR 2.5, 95% CI 1.4-4.5) and the difference remained significant when analysis was adjusted for age (p=0.047), ASXL1 (p=0.003) or EZH2 (p=0.001) mutations. Similarly, compared to JAK2-mutated cases (n=309), survival was longer in patients with “type 1/type 1-like” (HR 0.4, 95% CI 0.3-0.5) but not in those with “type 2/type 2-like” (HR 0.9, 95% CI 0.5-1.6) CALR mutations; the difference in survival between JAK2 and “type 1/type 1-like” CALR mutated cases remained significant (P<0.01) when analysis was adjusted for age, ASXL1 or EZH2 mutations or DIPSS-plus score. Conclusions : CALR mutations in PMF might be subclassified into type 1-like and type 2-like variants, based on predicted helical propensity of their mutant C-terminus. The favorable impact of CALR mutations in PMF might be restricted to type 1 or “type 1-like” variants. Disclosures No relevant conflicts of interest to declare.

Clinicopathologic Analysis of Calr Mutation Subtypes in Myeloproliferative Neoplasms

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2822-2822 ◽  
Author(s):  
Yin Xu ◽  
Brian Kwok ◽  
Aine Yung ◽  
Rachel Flamholz ◽  
Zhao Wu ◽  
...  
Keyword(s):  
Platelet Count ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Disease Evolution ◽  
Cytogenetic Abnormalities ◽  
Calr Mutations ◽  
Marrow Cellularity ◽  
Calr Mutation

Introduction: The discovery of JAK2, MPL, and CALR mutations has significantly improved the diagnostic approach to BCR-ABL1-negative myeloproliferative neoplasms (MPN). Approximately 60% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) harbor a JAK2 or MPL mutation. CALR mutations account for the majority of the remaining cases, and are found in 50-70% of ET and 60-90% of PMF cases that are negative for JAK2 and MPL mutations. Most CALR mutations cause a 52-bp deletion (type 1) or a 5-bp insertion (type 2). These mutations are acquired early during disease evolution and activate JAK/STAT signaling. Prior studies have shown that CALR type 1 mutations are associated with a favorable impact on survival of PMF patients, but not those with ET. Some data also suggested that CALR type 2 mutations may be associated with unfavorable prognosis in PMF. To assess the clinicopathologic impacts of CALR mutation subtypes in ET and PMF, we evaluated a series of CALR-mutated cases and correlated subtypes of mutations with several clinical, laboratory, and genetic parameters. Methods: MPN cases positive for CALR mutations were retrieved from our database over a period of 14 months. CALR, JAK2, and MPL mutation analyses were performed by either fragment analysis with Sanger sequencing confirmation or Next-Generation sequencing. Chromosome analysis and FISH with probes for 5p15/5q31, 7p11/7q31, 8cen, 20q, and t(9;22) were performed in all cases. Other parameters obtained included age, gender, hemoglobin, WBC, platelet count, bone marrow blasts and histology, and JAK2/MPL mutation status. The data were analyzed with independent sample t-tests and a 2-tailed chi-square test. Results: A total of 100 consecutive cases of CALR mutated MPNs were identified, 86 of which had available marrow specimens for morphologic subclassification. We further studied the cohort of 86 cases, including 37 ET and 49 PMF patients. 49 were male and 37 female with a median age of 67 (range 31-88) years. 49 (57%) patients had type 1, 28 (33%) had type 2, and 9 (10%) exhibited other types of mutations. No JAK or MPL mutation was found in any cases. Among patients with type 1 mutations, 22 (46%) were ET and 27 (54%) were PMF. Type 2 mutations were seen in 9 (33%) ET and 19 (67%) PMF patients. Notably, 5 cases of ET with type 2 mutations displayed atypical megakaryocytic hyperplasia with variable size and tight aggregates. In contrast, ET with type 1 mutations generally exhibited large megakaryocytes with hyperlobated nuclei. Two cases of PMF with type 2 mutations had a remote history of ET and may represent myelofibrotic transformation. ET patients with type 2 mutations had lower marrow cellularity (mean: 40% vs. 57%; p=0.014) than those with type 1 mutations. There were no statistically significant differences in age, gender, average hemoglobin, WBC, platelet count, marrow blasts, or reticulin fibrosis between the two ET subgroups. While no significant differences in various parameters were observed between PMF patients with type 1 and type 2 mutations, type 2 mutations showed a trend toward a higher platelet count (mean: 714 K/uL vs. 513 K/uL; p=0.086). Chromosome abnormalities were seen in 12 cases (23%), including 11 cases of PMF and 1 case of ET. Among PMF cases, cytogenetic abnormalities were less frequently associated with type 1 mutation (3/27) than type 2 and other types of mutations (8/22) (6% vs. 36%; p=0.035). The number of cases with other types of CALR mutations was small (3 ET and 6 PMF); therefore, comparison of those cases with cases from type 1 or type 2 mutated groups was precluded. Conclusions: ET patients with type 2 mutations showed less marrow cellularity and more megakaryocytic abnormalities associated with PMF compared to those with type 1 mutations. Our observations may raise the question whether ET patients with type 2 CALR mutations are more likely to progress to post-ET myelofibrosis. Type 2 mutations were also associated with a higher platelet count and higher frequency of cytogenetic abnormalities in PMF. Thus, CALR type 2 mutations may have a greater impact on megakaryocytic hyperplasia and platelet count production. We hypothesize that CALR type 1 and type 2 mutations represent different disease subgroups with pathogenic and prognostic implications. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Contemporary Approach to CALR-Positive Myeloproliferative Neoplasms

2021 ◽  
Vol 22 (7) ◽  
pp. 3371
Author(s):  
Tanja Belčič Mikič ◽  
Tadej Pajič ◽  
Samo Zver ◽  
Matjaž Sever
Keyword(s):  
Myeloproliferative Neoplasms ◽  
Molecular Genetic ◽  
Diagnostic Algorithm ◽  
Primary Myelofibrosis ◽  
Cellular Mechanisms ◽  
Cellular Effects ◽  
Prognostic Importance ◽  
Calr Mutations

CALR mutations are a revolutionary discovery and represent an important hallmark of myeloproliferative neoplasms (MPN), especially essential thrombocythemia and primary myelofibrosis. To date, several CALR mutations were identified, with only frameshift mutations linked to the diseased phenotype. It is of diagnostic and prognostic importance to properly define the type of CALR mutation and subclassify it according to its structural similarities to the classical mutations, a 52-bp deletion (type 1 mutation) and a 5-bp insertion (type 2 mutation), using a statistical approximation algorithm (AGADIR). Today, the knowledge on the pathogenesis of CALR-positive MPN is expanding and several cellular mechanisms have been recognized that finally cause a clonal hematopoietic expansion. In this review, we discuss the current basis of the cellular effects of CALR mutants and the understanding of its implementation in the current diagnostic laboratorial and medical practice. Different methods of CALR detection are explained and a diagnostic algorithm is shown that aids in the approach to CALR-positive MPN. Finally, contemporary methods joining artificial intelligence in accordance with molecular-genetic biomarkers in the approach to MPN are presented.

scholarly journals Knock-Ins of Type-2 Calr Mutants Cause Myeloproliferative Neoplasm (MPN)-like Hematopoiesis in Mice

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2964-2964
Author(s):  
Keiji Minakawa ◽  
Koki Ueda ◽  
Osamu Nakajima ◽  
Tetsuro Yokokawa ◽  
Yusuke Kinishima ◽  
...  
Keyword(s):  
Research Funding ◽  
Gene Set Enrichment Analysis ◽  
Hematopoietic Stem ◽  
Cell Counts ◽  
Calr Mutations ◽  
Calr Mutation ◽  
Mouse Lines ◽  
Stat Pathway

MPNs, including polycythemia vera, essential thrombocythemia (ET) and myelofibrosis (MF), are characterized by proliferation of mature myeloid cells. A somatic mutation in a hematopoietic stem cell (HSC) that activates JAK/STAT pathway drives MPN. Following the JAK2V617F, the CALR insertion/deletion mutations (indels) are the second most frequent driver and present in 20-30% ET and primary MF. Two major indels, a 52-bp deletion (type 1, p.L367fs*46) and a 5-bp insertion (type 2, p.K385fs*47), account for 80% of the CALR mutations. In addition, there have been more than 100 other indels, which can be classified as type 1- and type 2-like mutations based on the length of negatively-charged amino acid (AA) stretch at the C-terminal side of mutated CALR. Patients with type 1/type 1-like CALR mutations exhibit more incidence of MF while type 2/type 2-like mutations are associated with higher platelet counts in ET (Petra et al, Leukemia, 2016). In previous studies, mice carrying type 1/type 1-like mutations, including knock-in (KI) models, showed mild ET- or MF-like hematopoiesis. Although wild-type (WT) CALR AA sequences are highly conserved between human and mouse, there have been no KI models of type 2/type 2-like CALR mutations. Here, we generated 2 lines of KI mice carrying type 2-like Calr mutations, 2-bp insertion (CR2i, p.K378fs*53) and 10-bp deletion (CR10d, p.K375fs*52), using the CRISPR/Cas9 method. Both KIs removed KDEL, altered AA charges and increased values for isoelectric point, which are similar to type 2/type 2-like mutations in MPN patients. Compared with WT mice, peripheral platelets (1277 ± 228 vs 1560 ± 344 x 109/L, p = 0.004) and leukocytes (14.4 ± 3.7 vs 18.7 ± 4.9 x 109/L, p = 0.006) were increased in CR10d mice, whereas blood cell counts were not different between CR2i and WT mice. In FACS, both CR10d (p = 0.04) and CR2i (p = 0.04) mice exhibited an increased myeloid-cell ratio in bone marrow (BM). Splenomegaly was not present, but histopathological study showed a significant increase and accumulation of large megakaryocytes in BM and spleen of both KI mouse lines. BM fibrosis was not present in any sample. Therefore, CR10d and CR2i mice mimicked ET-like and unclassifiable MPN-like hematopoiesis, respectively. Next, we studied the basis of MPN-like hematopoiesis in CR10d and CR2i mice. Colony forming-cell assay in the presence of cytokines showed reduced growth of CFU-E, especially in CR2i mice (p = 0.01) compared with WT mice, while there was no difference in growth of CFU-Mk between CR10d or CR2i mice and WT mice. TPO-independent colony growth was not observed in both KI mice. Correspondingly, FACS showed comparable expression of phospho-STAT3 (pSTAT3) in BM cells between CR10d or CR2i mice and WT mice in the absence of TPO. However, pSTAT3 was significantly upregulated both in CR10d and CR2i mice compared with WT mice in the presence of TPO, suggesting that high sensitivity of HSCs or progenitor cells to TPO contributes to MPN phenotype in these mice. Thus, we investigated HSC function by a competitive repopulation assay, in which we transplanted a mixture of BM cells from KI mice (Ly5.2) and Ly5.1 mice at a 1:1 ratio into lethally irradiated Ly5.1 mice, showed reduced repopulating capacity, especially in CR2i mice. In the second transplant recipients, cells derived from either CR2i or CR10d mice were markedly diminished, suggesting the reduced self-renewal capacity of an HSC carrying a type 2/type 2-like Calr mutation. Finally, we performed RNAseq for FACS-sorted HSC-enriched lineage-Sca1+Kit+ (LSK) cells, which revealed that approximately 70% of genes among differentially expressed genes were commonly upregulated or downregulated in CR2i and CR10d mice, suggesting a similarity in gene expression profile of LSK cells of these KI mouse lines. As a result, there were several pathways commonly affected in both CR2i and CR10d mice in gene set enrichment analysis, including upregulation of JAK/STAT pathway (FDRq = 0.060 in CR2i and 0.111 in CR10d). On the other hand, targets of polycomb recessive complex 2, which are important for HSC functions in MPNs (Ueda et al, Blood Adv, 2017), were downregulated in both KI mouse lines (FDRq = 0 in both CR2i and CR10d), possibly explaining the reduced repopulating capacities of CR2i and CR10d HSCs. In conclusion, our data indicate that type 2/type 2-like Calr mutation can cause MPN-like hematopoiesis. For disease progression, further mechanism may be required. Disclosures Yokokawa: Actelion Pharmaceuticals Ltd: Other: Donated Fund Laboratory. Ikeda:Kyokuto Pharmaceutical: Research Funding; Hokuyo Denki: Research Funding; Novartis Pharma: Honoraria; Takeda Pharmaceutical: Honoraria, Research Funding.

Mutations of Calreticulin in Myeloproliferative Neoplasms Contribute to Disease Progression Not through Inhibition of Phagocytosis, but through Enhanced Proliferation Production of Myeloid Myelo-Erythroid Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4594-4594
Author(s):  
Shinya Daitoku ◽  
Katsuto Takenaka ◽  
Takuji Yamauchi ◽  
Koichi Akashi
Keyword(s):  
Progenitor Cells ◽  
Myeloproliferative Neoplasms ◽  
Hematopoietic Cells ◽  
Jak2 V617f ◽  
Jak2 Mutation ◽  
Hematopoietic Stem ◽  
Calr Mutations ◽  
Phagocytosis Index ◽  
Calr Mutation

Abstract Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders characterized by overproduction of mature myeloid cells. Recently, somatic mutation of calreticulin (CALR) was frequently found in MPN patients who do not have JAK2 mutation. The CALR mutation in MPN patients usually resulted in loss-of-function of CALR, which may induce impairment of physiological phagocytotic pathway, because surface CALR plays a critical role for macrophages in recognition of low-density lipoprotein receptor-related protein 1 (LRP1) on the targets, mediating pro-phagocytic signals. We hypothesized that the non-functional CALR mutation renders cells resistant to phagocytosis, and impairs the “programmed cell removal” of progenitors or mature blood cells, resulting in accumulation of hematopoietic cells in MPNs. In 135 Japanese MPNs patients enrolled in this study, including polycythemia vera (PV), essential thrombocytosis (ET) or primary myelofibrosis (PMF), 34 patients (25.2%) had CALR mutations, and 80 (59.3%) patients had JAK2 V617F mutation, respectively. CALR mutations were heterozygous in all 34 patients (27 patients with ET, 7 with PMF). On the other hand, JAK2 V617F mutations were found in 26 patients with PV, 39 with ET, and 15 with PMF. The expression levels of pro-phagocytotic CALR were normal in these MPN patients. We then performed in vitro phagocytosis assay to test whether the heterozygous CALR mutation affects engulfment of blood cells by macrophages. Hematopoietic stem cells (HSCs), progenitor cell populations such as common myeloid progenitors (CMPs), megakaryocyte/erythroid progenitors (MEPs) and granulocyte/monocyte progenitors (GMPs), and mature myeloid cells were isolated and opsonized, and were co-cultured with activated macrophages for 2 hours. After the culture, we enumerate macrophages and engulfed cells to analyze phagocytosis index (number of engulfed cells/number of macrophages) (Kuriyama et al. Blood 2012). However, the phagocytosis index was not changed in any of purified hematopoietic cells, irrespective of the presence of CALR or JAK2 mutation. These results strongly suggest that heterozygous, non-functional CALR mutation, and gain-of-function JAK2 mutations should not affect the engulfment process for hematopoietic cells by macrophages. We then investigated the effect of CALR or JAK2 mutations on differentiation and proliferation of stem or progenitor cells in MPNs. We performed colony-forming cell assay of multipotent cells, such as HSCs and CMPs, and evaluated clonal burden of CALR and JAK2 mutations in colonies derived from these stem and progenitor cells. In vitro culture showed that HSCs and CMPs with CALR and JAK2 mutations gave rise to granulocyte/monocyte (GM) or megakaryocyte/erythroid (MegE)-related colonies, whose frequencies were almost identical to those in wild-type controls, suggesting that these mutations do not affect myelo-erythroid lineage commitment at the multipotent stem or progenitor stages. In contrast, when we cultured GMPs and MEPs, frequencies of colonies with CALR or JAK2 mutations were significantly higher as compared to those in HSCs or CMPs (P<0.05); In patients with CALR mutation, 32.5% of HSC-derived colonies had CALR mutations, whereas in MEPs and GMPs, CALR mutations were found in 51.0% and 70%, respectively. In JAK2 mutated MPNs, 17.2% of HSC-derived colonies had JAK2 mutation, whereas 64.7% of MEP- and 87.9% of GMP-derived colonies had this mutation. These results indicate that clones with CALR or JAK2 mutations could contribute more robustly to maintain MEPs and GMPs, and these committed progenitors with mutations might produce higher amounts of mature myelo-erythroid cells, leading to progression of MPNs. Thus, CALR mutation contributes to progression of MPN, not through inhibition of phagocytic clearance, but presumably through enhanced production of myelo-erythroid lineage cells, as JAK2 mutation does. It is important to investigate the mechanism on which the CALR mutation causes overproduction of myelo-erythroid cells in future study. Disclosures No relevant conflicts of interest to declare.

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