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.

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.

A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5215-5215
Author(s):  
Munazza Rashid ◽  
Rifat Zubair Ahmed ◽  
Shariq Ahmed ◽  
Muhammad Nadeem ◽  
Nuzhat Ahmed ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Diagnostic Algorithm ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Common Mutation ◽  
Hypochondriac Region ◽  
Exon 9 ◽  
Calr Mutations

Abstract Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F. JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U. In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern. Disclosures No relevant conflicts of interest to declare.

A Heterogeneous Response Pattern to Interferon-alpha2 with Induction of a Significant Decrease in the Calreticulin Mutant Allele Burden in a Subset of Patients with Essential Thrombocythemia and Primary Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4057-4057
Author(s):  
Sabrina Cordua ◽  
Lasse Kjaer ◽  
Morten Orebo Holmström ◽  
Niels Pallisgaard ◽  
Vibe Skov ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Mutant Allele ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
Allele Burden ◽  
Travel Grant ◽  
Ifn Treatment

Abstract Introduction The discovery of mutations in the calreticulin (CALR) gene in the majority of JAK2 -V617F negative patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) (Klampfl et al., 2013; Nangalia et al., 2013) has improved the diagnostic accuracy considerably, and most recently distinct clinical and hematological characteristics according to mutational status have been described (Park et al., 2015). The perspective is to personalize and optimize treatment according to the molecular and clinical landscape. This may be achieved by obtaining more information on responses in myeloproliferative neoplasms (MPN) to existing treatment strategies as assessed by the allele burden. Mutations in the CALR gene have proven to play a major role in oncogenic and immunologic processes (Lu, Weng, & Lee, 2015). In this context, it is highly relevant to explore the effectiveness of interferon-alpha2 (IFN) in reducing the CALR -mutated clone. Until now, only one paper has reported a decrease in allele burden in two patients during IFN treatment (Cassinat, Verger, & Kiladijan, 2014). The objective of this report is to expand current knowledge on this important topic by describing the mutant CALR allele burden over time in a larger group of IFN-treated patients. Method Clinical data were collected retrospectively from a single institution on all IFN-treated CALR positive MPN patients with sequential determinations of the mutant allele burden. Type 1 and type 2 mutations were initially identified by a previously published fragment analysis (Klampfl et al 2013). We have developed a Taqman qPCR assay for precise determination of the mutant allele burden of type 1 and type 2 mutations. Stored DNA was subsequently analysed to increase follow-up time. Results Twenty-one patients were included. Fifteen patients had a diagnosis of PMF; 7 of these were diagnosed with prefibrotic myelofibrosis. Six patients had ET. The type 1 and 2 mutations were found in 15 and 6 patients, respectively. Median age was 60 years (range 42-79) and the sex ratio (M/F) was 8/13. Fifteen patients (71%) were in ongoing treatment with IFN, whereas treatment was discontinued in 6 (29%) because of side effects. Median time of IFN treatment was 756 days (range 42-3927). The IFN prescribed was either subcutaneous injection of Pegasys® (median: 45 microgram (ug) per week), PegIntron® 25-50 ug per week, or Multiferon® 3 x 3 million IU per week. Median follow up time since the first CALR measurement was 756 days (range 294-2108). Fourteen patients (67%) maintained an unchanged allele burden during follow up; 1 patient (5%) presented a temporary decrease (from 39% to 27% in allele burden) but increased to the initial level within months while still on IFN treatment (presumably due to low compliance); 1 patient (5%) displayed an increase in allele burden during transformation to acute myelogenous leukemia (Figure 1); and 5 patients (24%) exhibited a marked decrease in allele burden (median decrease: 32%, range 18-45) during treatment with IFN (Figure 2). All 5 patients with decreasing allele burden (Table 1) normalized their platelet counts within a median time of 5 weeks (range 4-20) after initiating treatment with IFN. Conclusion Using a novel sensitive assay for the CALR mutant allele burden, we have demonstrated and substantiated the effectiveness of IFN to reduce the allele burden in a larger series of CALR positive patients with PMF and ET. Importantly, we report for the first time on highly heterogeneous response patterns. Our observation of one fourth of the CALR positive patients responding to treatment with IFN strongly suggests that IFN significantly influences the CALR mutational load. Further clinical and molecular studies are urgently needed to explore the mechanisms behind the heterogeneous response patterns and the clinical implications in regard to clonal evolution and disease progression in non-responding patients. We are currently analysing these issues to assess the definite role of IFN in future treatment strategies in CALR positive MPN patients. Table 1. Patients responding to interferon-alpha2 Characteristics Number/median (range) Patients 5 Age, years 53 (42-62) Sex (M/F) 1/4 Diagnosis- Essential thrombocythemia- Primary myelofibrosis- Prefibrotic myelofibrosis 221 Calreticulin mutation type- type 1- type 2 50 Duration of interferon-alpha2 treatment, days 960 (177-2790) Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Cordua: Janssen-Cilag: Other: travel grant. Off Label Use: interferon alpha2 for myeloproliferative neoplasms. Holmström:La Roche Ltd: Other: travel grant. Pallisgaard:Qiagen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Speakers Bureau; Bristol Meyer Squibb: Speakers Bureau; Novartis: Other: travel grant, Research Funding, Speakers Bureau; Roche: Other: travel grant. Hasselbalch:Novartis: Research Funding.

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.

Calr Is up-Regulated in Patients with Essential Thrombocythemia Independently from Calr and JAK2 Mutations

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5581-5581
Author(s):  
Lilia Brown ◽  
Ciaren Graham ◽  
Ciro Rinaldi
Keyword(s):  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Over Expression ◽  
Molecular Abnormalities ◽  
Mutational Status ◽  
Significant Difference ◽  
Exon 9 ◽  
Calr Mutations

Abstract Somatic mutations in exon 9 of calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations, and absent in patient with polycythemia vera (PV). Among patients with ET or PMF with un-mutated JAK2 or MPL, CALR mutations were detected in 67% of those with ET and 88% of those with primary PMF. Several types of insertions or deletions were identified and all resulted in a frameshift in exon 9 generating a novel C-terminal peptide in the mutant CALR protein. Over expression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with myeloproliferative neoplasms carrying CALR mutations present with higher platelet counts and lower haemoglobin levels than patients with mutated JAK2. Studies suggest these patients have a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. We analysed by Real Time PCR, CALR expression in peripheral blood (PB) of 38 patients affected by ET, 17 JAK2 mutated (45%), 4 CALR (10.5%) mutated, 1 MPL mutated (3%) and 14 with no apparent molecular abnormalities. These were compared with a cohort of healthy volunteers. We found a significant over expression of CALR (median 5.15; range 1.13-270.08) comparing with controls (median 0.38, range 0.18-1). CALR mRNA expression is independent from the CALR mutational status. No significant difference was found comparing CALR expression in CALR mutated (median 4.9, range 1.51-37.14) and CALR/JAK2 un-mutated patients (4.68, range 1.51-28.71). CALR up-regulation is not mutually exclusive with JAK2 mutations; no difference was seen in CALR mRNA between JAK2 mutated (median 5.09, range 1.13-270) and wild type ET patients (median 5.08, range 1.51-37). There was no significant difference when we correlated CALR expression with PLT counts, spleen size or type of cytoreductive therapy. A larger cohort of patients is required to confirm these preliminary findings. Disclosures No relevant conflicts of interest to declare.

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.

Calreticulin Mutation Is Associated with Milder Disease in Patients with Post Essential Thrombocythemia Myelofibrosis (PET-MF) Compared with JAK2V617F Mutation: A Study from the AGIMM Group

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3179-3179
Author(s):  
Paola Guglielmelli ◽  
Giada Rotunno ◽  
Giada Brogi ◽  
Annalisa Pacilli ◽  
Costanza Bogani ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Allele Burden ◽  
Risk Of Death ◽  
Kaplan Meier ◽  
Significant Difference ◽  
Calr Mutations ◽  
The Impact ◽  
Calr Mutation

Abstract Background: Mutations in the gene calreticulin (CALR) were recently discovered in 60-80% of patients (pts) with primary myelofibrosis (PMF) and essential thrombocythemia (ET) who were un-mutated for JAK2V617F and MPLW515. CALR mutated PMF pts had better overall survival (OS) compared with JAK2V617F or MPLW515 mutated while in ET CALR mutations were associated with lower incidence of thrombosis although the effect on survival was not significant. Conversely, there is no information concerning the impact of CALRmutation on disease phenotype and prognosis in post-essential thrombocythemia myelofibrosis (PET-MF). Aims: The aim of the study was to assess whether CALR mutational status and/or allele burden had clinical and/or prognostic relevance in PET-MF compared with JAK2, MPLmutated or triple-negative (TN) pts. Methods: ET and PET-MF were diagnosed by 2008 WHO and IWG-MRT criteria respectively; all pts provided an informed consent. Genotyping for CALR, JAK2V617F and MPLW515 was performed in granulocytes using allele specific RTQ-PCR (JAK2, MPL), capillary electrophoresis and direct sequencing (CALR, MPL). The prognostic value of the molecular variables with regard to OS was estimated by the Kaplan-Meier method and Cox regression. Results: A series of 147 PET-MF pts from 4 Italian centres was collected. Pts median age was 63y. Median follow up from PET diagnosis was 3.2y (0.07-18.8y) and the median time from ET to PET diagnosis was 11.6y (0.9-30.6y). Death occurred in 38 pts (26%) and 14 pts (9.5%) developed acute leukemia (AML). The median OS in the entire series calculated from PET-MF diagnosis was 10.9y (7.1-14.7y). Frequency of mutations was: CALR 16%, JAK2V617F 77%; MPLW515 4.3%; TN 2.8%. The frequency of CALR mutations in PET-MF patients was superimposable to that observed in a control group of 576 ET patients from our Institution (15.5%) and slightly lower compared with other series (20-25%). Type of CALRmutations was: 59.6% type 1, 23.1%, type 2, 17.3% others, significantly different (P=0.023) from ET: 46% type 1, 38% type 2, 16% others. Median CALR allele burden in PET-MF was 56% (20%-100%) with no significant differences in the CALR mutation subtypes (57.5% in type 1, 47.5% in type 2 and 45.0% in others); however, the median mutant allele burden of CALR-mutated PET-MF patients was significantly higher than in ET patients (33%, range 2%-52%; n=100) (P<0.03) suggesting a role for mutated allele accumulation in evolution to PET-MF. Similarly, the median V617F allele burden in JAK2 mutated patients was 50.5% (range 5-100%) significantly greater than the value (24%; range, 1-87%) (P=0.02) in ET pts, confirming previous data that evolution to PET-MF is associated with accumulation of mutated JAK2allele. We then compared hematological and clinical characteristics of the patients who were categorized according to their JAK2V617F, MPLW515 and CALR mutation status. There was no statistically significant difference among the unique patient mutational groups regarding age, hemoglobin, leukocyte and platelet count, peripheral blasts, LDH, circulating CD34+ cells, abnormal karyotype, grade of bone marrow fibrosis and cellularity, and pruritus. However, JAK2+ pts showed an increased rate of large (>10 cm) splenomegaly (28.6% vs 14% in CALR+, 7.1 in MPL+ and 25% in TN pts; P=0.02) and constitutional symptoms (50% vs 18.8% in CALR+, 45% in MPL+ and 12.5% in TN pts; P=0.002). The interval from ET to PET-MF was significantly longer in CALR+ pts (14.5y) compared with JAK2+ (10.2y) and TN patients (11.0y; P=0.04 for both) and similar to MPL+ (14y). There was a reduced rate of death (13.5%) in CALR+ compared with JAK2+ (30.6%), MPL+ (21.4%) and TN (66.7%) pts (P=0.005), although Kaplan Meier estimates did not reach a statistically significant difference. Finally, there were less AML transformation in CALR+ pts (1.9%) compared with JAK2+ (13.9%), MPL+(7.1%) and TN (22.2%) (P=0.04). Conclusion: These results show that CALR mutation is associated with delayed transformation of ET to PET-MF, a milder disease in terms of splenomegaly and symptom burden and a reduced risk of death compared with JAK2V617F PET-MF pts and more in general with MPL mutated and TN pts.In addition, similar to findings in primary MF and unlike in ET, PET-MF is characterized by prevalence of type 1 CALR mutations. 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.

Calreticulin Mutations Variants of ET and PMF Patients Studied in Almazov Medical Research Center Russian Federation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5499-5499 ◽  
Author(s):  
Pavel Butylin ◽  
Natalia Matyuhina ◽  
Ekaterina Lisina ◽  
Nadia Siordia ◽  
Olga Senderova ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Sequence Similarity ◽  
Mutation Ratio ◽  
Exon 9 ◽  
Calr Mutations ◽  
Group Sex ◽  
Type 3 ◽  
Mutation Type

Abstract We analyzed CALR mutations in cohort of patients with ET and PMF. Diagnoses were determined according to the WHO criteria. 33 PMF and 45 ET patients that were negative for JAK2V617F and MPL515L/K mutations have been studied for CALR mutations presence as described in original paper (Т.Klampfi , 2013). 29 ET and 25 PMF patients found mutated. In PMF group sex ratio was equal - 12 males/13 females, in ET group females prevailed - 21 males/69 females. Median age in PMF group was 64 (41-78) and in ET group 58 (22-80). Exon 9 was Sanger sequenced and mutation type identified. All the mutated CALR sequences were translated with verified +1 frameshift with alternative C-end. In PMF group we found type 1 52bp deletion p.L367fs*46 (n=11), type 2 TTGCT insertion p.K385fs*47 (n=5), type 3 p.L367fs*48 (n=3), type 7 p.K368fs*45 (n=2), type 19 p.E372fs*48 (n=2) and single cases of p.K368fs*51, p.K385fs*47 , p.Q365fs*50. In ET group we found type1 52bp deletion p.L367fs*46 (n=10), type 2 TTGCT insertion p.K385fs*47 (n=6), and single cases of p.L367fs*52, p.K368fs*51, p.E378fs*45, p.K374fs*49), p.E364fs*49. Also in ET group we identified two cases with alternative insertion c.1154_1155insGTGTC p.E386fs*46. In one ET case we found complex CALR mutation variant c. с.1126_1142delInsACCTTTGCATTTT (p.R376fs*53). In this case alternative C-end, different from what was described before will be formed. CALR mutations were grouped in Type1 like and Type2 like according to protein sequence similarity (A. Tefferi, 2014). In PMF group type1/2 like mutation ratio was 18/7 and in ET group it was 16/13, confirming higher CALR type2 like mutations rate in ET. We analyzed CALR mutations in cohort of patients with ET and PMF. Diagnoses were determined according to the WHO criteria. 33 PMF and 45 ET patients that were negative for JAK2V617F and MPL515L/K mutations have been studied for CALR mutations presence as described in original paper (Т.Klampfi, 2013). 29 ET and 25 PMF patients found mutated. In PMF group sex ratio was equal - 12 males/13 females, in ET group females prevailed - 21 males/69 females. Median age in PMF group was 64 (41-78) and in ET group 58 (22-80). Exon 9 was Sanger sequenced and mutation type identified. All the mutated CALR sequences were translated with verified +1 frameshift with alternative C-end. In PMF group we found type 1 52bp deletion p.L367fs*46 (n=11), type 2 TTGCT insertion p.K385fs*47 (n=5), type 3 p.L367fs*48 (n=3), type 7 p.K368fs*45 (n=2), type 19 p.E372fs*48 (n=2) and single cases of p.K368fs*51, p.K385fs*47 , p.Q365fs*50. In ET group we found type1 52bp deletion p.L367fs*46 (n=10), type 2 TTGCT insertion p.K385fs*47 (n=6), and single cases of p.L367fs*52, p.K368fs*51, p.E378fs*45, p.K374fs*49), p.E364fs*49. Also in ET group we identified two cases with alternative insertion c.1154_1155insGTGTC p.E386fs*46. In one ET case we found complex CALR mutation variant c. с.1126_1142delInsACCTTTGCATTTT (p.R376fs*53). In this case alternative C-end, different from what was described before will be formed. CALR mutations were grouped in Type1 like and Type2 like according to protein sequence similarity (A. Tefferi, 2014). In PMF group type1/2 like mutation ratio was 18/7 and in ET group it was 16/13, confirming higher CALR type2 like mutations rate in ET. Klampfl T. et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013 Dec 19;369(25):2379-90. Tefferi A. et al. The prognostic advantage of calreticulin mutations in myelofibrosis might be confined to type 1 or type 1-like CALR variants. Blood. 2014 Oct 9;124(15):2465-6. Disclosures Zaritskey: Janssen: Consultancy; Novartis: Consultancy.

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.

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