Differential Association of Calreticulin Type 1 and Type 2 Mutations with Myelofibrosis and Essential Thrombocytemia: Relevance for Disease Evolution

Abstract Recent advances in myeloproliferative neoplasms (MPN) have highlighted the prevalence of mutations in the calreticulin gene (CALR), bringing a major new actor in these disorders. CALR mutations were reported in 25% of ET and in 35% of MF patients, which were non-mutated for JAK2 and MPL. CALR mutations lead to a frame-shift generating a common 36 amino acids C-terminal end and loss of the KDEL motif. Two variants account for 85% of the CALR mutations in ET and PMF: type 1, a 52-bp deletion and type2, a 5-bp insertion. 572 MPN patients negative for JAK2 and MPL mutations were collected from several French and Belgian hospitals. In our series, 396 patients were diagnosed as ET, 108 as MF and 68 as mixed MDS/MPN. We identified mutations of CALR in 368 patients (63.3%). The remaining 204 patients were designated as triple negative. In MF there was an over representation of type 1 mutation (70%) and an under representation of type 2 mutation (13%) as compared to patients with ET. This bias was associated with a higher allelic burden of CALR mutation in MF. MF patients represent a quite homogeneous group, mostly composed of men diagnosed at a median age of 62.5 with a low hemoglobin concentration (10.1 g/dl) and a low platelet count (median at 237 x 109/l). In ET patients the clinical presentation was more heterogeneous. They were mostly women (more than 61%) at a median age of diagnosis of 57 with a median platelet count of 724 x 109/l. In CALR mutated patients there were no sex prevalence and a more important thrombocytosis (785 x 109/l). The type of CALR mutation impacted also age and platelet count. We report the caracterisation of triple negative patients. In ETs they were mostly women (76.9%), particularly for ET patients under 50 years old that were almost exclusively women (27/28). In MF, triple negative patients presented a low hemoglobin concentration (8.85 g/dl) and a low leukocyte count (1.995 x 109/l). A striking characteristic is their platelet count, which was significantly lower than their group mates either in ET or in MF. This lower platelet count may suggest that in the general population, putative asymptomatic triple negative ET male patients could be retrieved, which would only be diagnosed at more advanced age with a symptomatic MF. Taken together, our results underline the differences between the two most frequent types of CALR mutation and show that CALR mutated patients should not be considered as a single entity. Disclosures No relevant conflicts of interest to declare.

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Clinicopathologic Analysis of Calr Mutation Subtypes in Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v126.23.2822.2822 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2822-2822 ◽

Cited By ~ 1

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.

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Calreticulin mutations Are Present in Polyclonal As Well As Clonal ET

Blood ◽

10.1182/blood.v124.21.4590.4590 ◽

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.

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

Blood ◽

10.1182/blood.v124.21.3215.3215 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3215-3215 ◽

Cited By ~ 1

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.

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A NEW TYPE-2-LIKE CALR MUTATION IN ESSENTIAL THROMBOCYTHEMIA

International Journal of Case Reports ◽

10.28933/ijcr-2021-02-1805 ◽

2021 ◽

pp. 204

Author(s):

Keyword(s):

Myeloproliferative Neoplasms ◽

Driver Mutations ◽

Chronic Myeloproliferative Neoplasms ◽

Younger Age ◽

New Type ◽

Leukocyte Counts ◽

Calr Mutations ◽

Calr Mutation

CALR mutations, together with JAK-2 and MPL ones, are recognized as “driver” mutations in Philadelphia-negative chronic myeloproliferative neoplasms (MPNs). Most frequent CALR mutations are Type-1 deletions (45-55% of cases) and type-2 insertion (32-42% of cases). These mutations are usually associated with younger age, higher platelet counts, lower leukocyte counts, lower hemoglobin levels and a higher incidence of transformation from ET to MF. Recognizing and describing cases with different mutations can be useful to create a database that might help clinicians to include these patients in risk categories and to guide the appropriate therapeutic choices. We report a case of a 77-years old woman who presented a new type-2 like CALR mutation.

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Driver Mutation Profile By MLPA in Myelofibrosis Patients

Blood ◽

10.1182/blood-2018-99-119776 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5494-5494

Author(s):

Minuncio Juliana ◽

Alexandre Nonino ◽

Juliana Forte Mazzeu ◽

Cintia do Couto Mascarenhas

Keyword(s):

Bone Marrow ◽

High Risk ◽

Acute Leukemia ◽

Triple Negative ◽

Low Risk ◽

Calr Mutations ◽

Mutation Type ◽

Calr Mutation

Abstract Myelofibrosis is the rarest and most severe Ph- myeloproliferative neoplasm and can present de novo or post Polycythemia Vera or Essential Thrombocythemia. It is characterized by bone marrow fibrosis, extramedullary hematopoiesis and abnormal expression of inflammatory cytokines resulting in several atypical events and may progress to Acute Leukemia. The disease arises from clonal expansion of a single hematopoietic stem cell (HSC), driven by a somatic mutation of JAK2, CALR or MPL genes combined with dysregulation of hematopoietic microenvironment, additional mutations and cytogenetic abnormalities.The aims of this study are to assess driver mutations status in primary or secondary myelofibrosis patients and to correlate their mutational profile with clinical outcomes. The search for JAK2V617F, exon 12 JAK2, calreticulin exon 9 c.1092_1143del52 and c.1154_1155insTTGT, MPLW515K and MPLW515L mutations was performed in 31 subjects using MLPA technique, a method of DNA analysis that allows simultaneous appraisal of different mutations in multiple samples. 48.4% of patients present the JAK2V617F mutation,indel CALR mutations in 38.7% of patients (of these, 66.7% with del52 bp, 33.3% harbored insTTGTC),MPL W515L in 3.2% of patients and 9.7% of patients were triple-negative. From the mutational profile information obtained by MLPA,the clinical-molecular risk score was calculated for each of the individuals in the sample, according Rumi et al.. Being at 12,9% (4) as very low risk, 9.7% (3) as low risk,35.4% (11) as intermediary risk, 29.1% (9) and of high risk, and 12.9%(4) as very high risk. Patients with mutated JAK2 were older, with minor degree of anemia and more leukocytosis, whereas those with CALR mutations had less frequency of leukocytosis and thrombocytopenia. Triple-negative subjects displayed the lowest median age at diagnosis (49.3 years), and bone marrow failure phenotype, similar to Myelodysplastic Syndrome. Risk stratification provided by DIPSS was similar to other centers.Individuals with PMF present constitutional symptoms significantly more often than those with post-ET MF(p = 0.0365).Mortality rate was 29%, and mean survival after diagnosis was 68.3 months. CALR mutated individuals presented higher average survival and median survival according to DIPSS was higher than predicted by the prognostic model, possibly due to the higher frequency of CALR mutations reported.Median follow-up time was 32 months (ranging from 10 months to 13 years).Thromboembolic phenomena were recorded in 19.3% of patients, and evolution to AML in 6.4% of patients and it was verified that 75% of the individuals with Myelofibrosis Post-ET presented thrombotic events at some point in the disease. The association between the DIPSS clinical-laboratory parameters and the demand of transfusion at diagnosis, with the occurrence of Acute Leukemia was assessed using Fisher's exact test but have no significant difference in these parameters between patients who evolved or not for Acute Leukemia. The JAK2 V617F mutation is expected to be present in 60 to 65% of individuals with Myelofibrosis, but this mutation has been identified in only 48% of patients. In contrast, the observed frequency of indel of the CALR, of 38%, was higher than the classically described, from 25 to 30%. The rate of mutation type 2 (ins 5-bp) was also higher than expected, 33.6%.Regarding the mutation subtypes of CALR, mutation type 1 (52-bp deletion) is observed in up to 80% of MFP cases, but has a similar frequency as type 2 (5-bp insertion) in patients with ET. The grouping of patients with primary MF and post-ET may have contributed to the higher incidence of type 2 CALR mutation observed in this sample, although much higher than the 13% frequency described in the literature in a mixed population. The type 1 mutation was observed in 66% of our patients with post-ET and mutated CALR, but the small number of individuals in the study does not allow to estimate the impact of this mutation in the evolution of the disease. Our fraction of CALR-mutated patients is much higher than those described in Asian, European, North American and Argentinean populations. The complex genetic landscape involved in initiation and progression of Myelofibrosis, instigates the adoption of integrative prognostic stratification models. In this scenario, MLPA is a powerful tool for molecular study, and a promising ally for MPN molecular characterization. Disclosures No relevant conflicts of interest to declare.

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Type I Calreticulin Mutations Result in Hyperactivation of Its Acetyltransferase Function and Iron Metabolism, Inducing a Susceptibility to Ferroptosis

Blood ◽

10.1182/blood-2021-153480 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3593-3593

Author(s):

Harrison S Greenbaum ◽

Maria Evers ◽

Alex Rosencrance ◽

Luke Maxwell ◽

Katarzyna Kurylowicz ◽

...

Keyword(s):

Iron Metabolism ◽

Transferrin Receptor ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Type I ◽

Lipid Peroxidation Product ◽

Calr Mutations ◽

The Impact

Abstract Approximately 20% of patients with myeloproliferative neoplasms (MPN) harbor mutations in the gene calreticulin (CALR). Of these, approximately half are classified as type 1 and 30% as type 2, characterized by a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5) respectively. Although both share identical mutant C-termini and are able to bind and activate MPL, type 1 and type 2 CALR mutations display different clinical and prognostic presentation: type 1 mutations are associated primarily with a fibrotic phenotype and increased proclivity towards fibrotic transformation, while type 2 mutations are more common in ET. Molecularly, type 1 and type 2 mutations result in differential C-domain amino acid sequences with the potential to affect the function of the protein. Various well known functions of CALR, including calcium binding ability and protein folding capacity, have begun to be explored in the context of CALR mutations; however, the impact of CALR mutations on its acetyltransferase ability, which was only discovered in 2006, remains unknown. Here, we show that in accordance with our structural models, mutant CALR not only retains its acyltransferase ability, but type 1 CALRdel52 mutations specifically lead to increased activation of its acetyltransferase ability, revealing a new gain of function phenotype for CALRdel52 mutations. As a result, type 1 CALR mutations lead to increased acetylation of CALR's acetyltransferase targets downstream, such as glutathione-S-transferase and cytochrome P450 reductase, which affects the outputs of these pathways downstream. Exploratory RNA-Seq on CALR-mutated cells revealed a concurrent upregulation of transferrin receptor mediated iron metabolism by CALRdel52. We subsequently validated this finding and show that CALRdel52 cells display differential iron metabolism. Given the upregulation of the transferrin receptor and the increased acetyltransferase ability affecting proteins involved in reactive oxygen species pathways (ROS), ferroptosis-an iron-dependent form of cell death characterized by the accumulation of lipid peroxides-emerged as a potential therapeutic target for CALRdel52 mutated cells. To test this, we first assessed basal proclivity to ferroptosis by measuring the lipid peroxidation product, classic ferroptotic marker 4-HNE (4-hydroxynonenal) as well as both ROS and global lipid peroxide levels in cells expressing wild-type CALR, CALRdel52, and CALRins5. We found that all of these ferroptotic markers were significantly increased in CALRdel52 cells. Therapeutic modulation of these pathways such as iron supplementation resulted in targeting of CALRdel52 cells and ferroptosis induction. This work is the first to examine the acetyltransferase ability of mutant CALR and reveal downstream phenotypic differences based on this ability that set the groundwork for a host of unexplored cellular consequences. Moreover, this study unites the novel understanding of the acetyltransferase function of mutant CALR with changes in transferrin receptor mediated iron metabolism to reveal not only how CALRdel52 induces a ferroptotic proclivity, but the potential of this sensitivity for therapeutic targeting. Disclosures No relevant conflicts of interest to declare.

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Outcome of Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis (RARS-T) In a Large Cohort of Patients

Blood ◽

10.1182/blood.v116.21.4113.4113 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4113-4113 ◽

Cited By ~ 1

Author(s):

Francois Girodon ◽

Julien Broseus ◽

Lourdes Florensa ◽

Esther Zipperer ◽

Susanne Schnittger ◽

...

Keyword(s):

Platelet Count ◽

Myeloproliferative Neoplasms ◽

Hemoglobin Concentration ◽

Large Cohort ◽

Refractory Anemia ◽

White Blood Count ◽

Employment Equity ◽

Disease Evolution ◽

Ringed Sideroblasts ◽

Equity Ownership

Abstract Abstract 4113 Introduction: Most of the data related to RARS-T, a rare disorder, involve small cohorts of patients. We aimed to analyze more patients also considering a variety of myelodysplastic or myeloproliferative disorders. Objective: To compare a large cohort of patients with RARS-T to refractory anemia with ringed sideroblasts (RARS), refractory anemia with ringed sideroblasts and multilineage dysplasia (RARS-MD) or essential thrombocythemia (ET) at the time of diagnosis and during disease evolution, in terms of survival and complications. Materials: Data of a European multi-center study was used including 199 cases of RARS-T 173 cases of RARS, 102 cases of RARS-MD and 431 cases of ET. Results: At baseline, compared to RARS and RARS-MD patients, RARS-T patients had similar hemoglobin concentration, but a higher white blood count. The JAK2V617F mutation was observed in 43%, 12% and 5% in RARS-T, RARS and RARS-MD patients, respectively. When separated in 2 groups (450,000<platelet count <600,000 and platelet count >600,000 × 109/l), RARS-T patients were comparable for sex, age, hemoglobin level and survival. However, patients with platelet count > 600,000 × 109/l had higher WBC (11 ×109/l versus 7.5 ×109/l, p<0.001). Similarly, no difference was noted in the survival in the JAK2 positive and negative RARS-T patients. The age and sex standardised overall survival of RARS-T patients was similar to RARS and RARS-MD patients, but lower than ET patients (p<0.001). This was despite a higher risk of transformation in acute leukemia, relative to RARS-T afflicted individuals, of 2.4 and 3.5 in RARS-MD and RARS patients, respectively. Conclusion: According to our results, the outcome in RARS-T more closely mimics myelodysplastic syndromes rather than myeloproliferative neoplasms. Our results agree with the WHO 2008 classification that considers RARS-T as a separate disorder. Disclosures: Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Gattermann:Novartis: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Driver Mutations and Prognosis in 502 Patients with Essential Thrombocythemia

Blood ◽

10.1182/blood.v126.23.1599.1599 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1599-1599 ◽

Cited By ~ 1

Author(s):

Yoseph Elala ◽

Terra L. Lasho ◽

Naseema Gangat ◽

Christy Finke ◽

A Kamel Abou Hussein ◽

...

Keyword(s):

Platelet Count ◽

Survival Data ◽

Triple Negative ◽

Univariate Analysis ◽

Multivariable Analysis ◽

Driver Mutations ◽

Mutational Status ◽

Age And Sex

Abstract Background : In essential thrombocythemia (ET) , ̴ 85% of patients harbor one of three "driver" mutations, with mutational frequencies of approximately 58%, 23% and 4%, for JAK2, CALR and MPL, respectively; ̴ 15% are wild type for all three mutations and are operationally referred to as "triple negative" (Blood. 2014;124:2507). In one of the original descriptions on CALR mutations, CALR -mutated patients with ET, compared to their JAK2-mutated counterparts, were reported to have better survival (NEJM. 2013;369:2379). However, this observation was not supported by subsequent studies while other reports suggested differential prognostic effect from distinct CALR variants in myelofibrosis (Blood. 2014;124:2465). In this study, we sought to clarify the impact of all three mutations, and CALR variants, on overall (OS), myelofibrosis-free (MFS) and leukemia-free (LFS) survival. Methods: Patientswere selected from our institutional database of myeloproliferative neoplasms, based on availability of mutational status inforomation. ET diagnosis was according to WHO criteria (Blood. 2009;114:937). Published methods were used for CALR, JAK2 and MPL mutation analyses and determination of CALR variants (Blood. 2014;124:2465). Kaplan-Meier survival analysis was considered from the date of diagnosis to date of death or last contact. MFS and LFS calculations considered fibrotic or leukemic transformation events as uncensored variables, respectively. Cox proportional hazard regression model was used for multivariable analysis. Results : A total of 502 patients (median age 59 year; 61% females) met study eligibility criteria. Median levels of hemoglobin, platelet count and leukocyte counts were 13.7 g/dL, 893 x 10 (9)/L and 8.8 x 10(9)/L, respectively. All patients were annotated for JAK2/CALR/MPL mutations as well as CALR variants; 324 harbored JAK2, 111 CALR and 13 MPL mutations; 54 patients were triple-negative. The 111 CALR-mutated patients included type 1 (n=55), type 2 (n=41) or other (n=15) CALR variants. At a median follow-up time of 9.9 years, 172 (34.3%) deaths, 42 (8.4%) fibrotic progressions, 15 (3%) blast transformations and 12 (2.4%) polycythemic conversions were documented. In univariate analysis, survival data appeared significantly better in "triple negative" patients (median not reached) and inferior in MPL-mutated cases (median 8.5 years) whereas median survival times were similar for JAK2 (18.5 years) and CALR (22.1 years) mutated cases (Figure 1; p=0.0006). However, the difference in survival was no longer apparent (p=0.60) during multivariable analysis that included age and sex, which are known to differentially cluster with specific driver mutations; in the current study, median age/sex distributions for "triple-negative", CALR, JAK2 and MPL mutated cases were 44 years/72% females, 48 years/46% females, 60 years/65% females, 70 years/46% females, respectively (p=<0.0001/0.0007). Of note, both age and sex were independently predictive of shortened survival. OS data remained unchanged when CALR-mutated patients were further stratified into type 1 vs type 2 vs other CALR variants, with similar survival data between the three CALR mutation groups (p=0.98). In univariate analysis, MPL-mutated patients were significantly more prone to fibrotic progression (Figure 2; p=0.0083). The prognostic relevance of MPL mutations to MFS remained significant when age and sex were included in multivariable analysis (p=0.008). In the current cohort, univariate analysis identified lower hemoglobin and lower platelet count as the only other risk factors for fibrotic progression. Multivariable analysis confirmed the independent prognostic relevance of MPL mutations (p=0.003), lower hemoglobin level (p=0.0009) and lower platelet count (p=0.0094) for MFS. There was no significant difference in LFS among the four driver mutational categories (p=0.9): 9 events in JAK2, 6 in CALR, none in triple negative and none in MPL mutated cases. Among the 6 leukemic transformations in CALR-mutated cases, three were type 1, two type 2, and one other CALR variants. Conclusions : Age- and sex-adjusted survival is similar among ET patients with JAK2 vs CALR vs MPL vs "triple-negative" mutational status. Survival is also similar between patients with distinct CALR variants. MPL -mutated patients with ET might be at a higher risk of fibrotic progression. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Pardanani: Stemline: Research Funding.

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Germline Calr Mutation and Thrombocytosis Presenting with Concomitant BCR-ABL1+ CML

Blood ◽

10.1182/blood.v128.22.5494.5494 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5494-5494 ◽

Cited By ~ 1

Author(s):

Linda B. Baughn ◽

Scott Gilles ◽

Elizabeth L. Courville ◽

Andrew C. Nelson ◽

Zohar Sachs

Keyword(s):

Platelet Count ◽

Allele Frequency ◽

Peripheral Blood ◽

Myeloproliferative Neoplasms ◽

Germline Mutations ◽

Myelogenous Leukemia ◽

Base Pair Deletion ◽

Exon 9 ◽

Calr Mutations ◽

Calr Mutation

Abstract CALR mutations are present in 70-84% of JAK2 wild-type myeloproliferative neoplasms (MPN) and 67% and 88% of essential thrombocytopenia (ET) and primary myelofibrosis (PMF) respectively. Most cases of MPN are apparently sporadic, but 7-11% have evidence of familial predisposition. While germline mutations in ET-associated genes, MPL and JAK2, have been described in hereditary thrombocytosis, germline mutations in CALR have not been described in any setting. Two types of CALR mutations are common in MPN: a 52-base pair deletion (bp) and a 5 bp insertion, both in exon 9. With rare exceptions, CALR mutations are generally mutually exclusive with JAK2 or MPL mutations and have very rarely been reported in conjunction with the BCR-ABL1translocation. Here, we report a patient with a germline CALR mutation, thrombocytosis, and subsequent development of BCR-ABL+ CML. A 67-year-old female with no significant medical history presented with severe abdominal pain and nausea. Peripheral blood analysis revealed a marked leukocytosis composed of 66% neutrophils, 16% myelocytes, 6.5% monocytes, 3.5% basophils, 2.5% promyelocytes, 2.5% metamyelocytes, 1.5% lymphocytes, 1.5% blasts, and no eosinophils. The patient was non-anemic and had a normal platelet count (340,000/mm3). Bone marrow biopsy revealed a hypercellular marrow with myeloid predominant trilineage hematopoiesis and 1-2% blasts with morphology consistent with chronic myelogenous leukemia (CML). Fluorescence in-situ hybridization analysis of peripheral blood identified a BCR-ABL1fusion in 98.5% of interphase cells. After 3 months of standard imatinib therapy, quantitative RT-PCR showed a reduction of BCR-ABL1/ABL1 in the peripheral blood, however platelet count was elevated at 539,000/mm3. Thrombocytosis persisted over 2 years with a maximal platelet count of 584,000/mm3. Given the patient's thrombocytosis, her peripheral blood was subjected to a next generation sequencing of JAK2, MPL, and CALR genes. A 52-bp out-of-frame deletion in exon 9 of the CALR gene was detected (52% allele frequency) in peripheral blood. In addition, the same 52-bp CALR deletion (63% allele frequency) was present at the time of diagnosis and within a buccal specimen (47% allele frequency) when the BCR-ABL1 transcript was 1% in the peripheral blood. Immunostain of the buccal sample was strongly positive for cytokeratin (CK) AE1/AE3 but CD45 was not detected indicating no leukocyte contamination. This case reports the first instance of a germline CALR mutation associated with thrombocytosis and is the fourth report of the co-occurrence of BCR-ABL1 and CALR mutation in a single patient. Evolution to BCR-ABL1+ CML suppressed the CALR-mutant thrombocytosis phenotype, emphasizing the effect of these genes on lineage determination in abnormal myeloid proliferation. Disclosures No relevant conflicts of interest to declare.

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Calr Mutational Status in Egyptian Patients with Myeloproliferative Neoplams

Blood ◽

10.1182/blood-2019-128194 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5388-5388

Author(s):

Eman A. Soliman ◽

Samah I. El-Ghlban ◽

Abdelaleem H. Abdelaleem ◽

Sherin Abdel-Aziz ◽

Sameh Shamaa ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasm ◽

Response To Therapy ◽

Hcv Infection ◽

Number Of Patients ◽

Significant Difference ◽

Egyptian Patients ◽

Calr Mutations

It has been known that the insertion/deletion mutation of CALR gene is the second deriver mutation in myeloproliferative neoplasm (MPN) of essential thrompocythemia (ET) and primary myelofibrosis (PMF). As the molecular workup has been incorporated for the prospective screening and diagnosis of MPN in our Oncology Center. An Egyptian 87 cohort of patients with non-mutated JAK2 (58 ET and 29 MF) were investigated using polymerase chain reaction (PCR) as a pilot study. We found that 37 out of 87 patients (42%) were carrying CALR mutations (30 out of ET (52%) and 7 out of MF (24%)). Sanger sequencing was used to determine the type of CALR mutations in all positive patients and we found that 13 out of 37 (35%) had type 1/type 1-like and 36 out of 37 (97%) with type 2/type 2-like. This CALR mutation profile in Egyptian patients appear different from the western status as type2/type 2-like is the highest in our patients (97%) versus 35-45% and type1/type 1-like was 35% versus 55-65% compared to western results. Meanwhile, the clinical course and phenotype of our cohort of patients is not similar to that in western as there is no significant difference of overall survival between type1/type1-like and type2/type2-like. This finding might be due to the different environmental and genetic backgrounds of Egyptian population. A part of it might be related to the HCV infection as 12 out of 37 (32%) had HCV infection. Further study is in progress on a large number of patients to correlate that with the clinicopathological status, response to therapy and the mechanistic pathway of oncogenic transformation. Disclosures No relevant conflicts of interest to declare.

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