Driver Mutation Profile By MLPA in Myelofibrosis Patients

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

Blood ◽

10.1182/blood.v124.21.1823.1823 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1823-1823 ◽

Cited By ~ 1

Author(s):

Xenia Cabagnols ◽

Jean-Philippe Defour ◽

Valérie Ugo ◽

Jean Christophe Ianotto ◽

Pascal Mossuz ◽

...

Keyword(s):

Platelet Count ◽

Triple Negative ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Hemoglobin Concentration ◽

Disease Evolution ◽

Calr Mutations ◽

Calr Mutation

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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Study on Methylation of p15INK4B Gene and Its Mechanisms in Patients with Myelodysplastic Syndrome and Leukemia.

Blood ◽

10.1182/blood.v106.11.3441.3441 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3441-3441

Author(s):

Hongyan Tong ◽

Maofang Ling ◽

Jie Jin

Keyword(s):

Bone Marrow ◽

High Risk ◽

Acute Leukemia ◽

Myeloid Leukemia ◽

Mononuclear Cells ◽

Chronic Phase ◽

Lymphocytic Leukemia ◽

Low Risk ◽

Blast Phase ◽

Genetic Event

Abstract The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in the mononuclear cells (MNCs) from bone marrow of 54 cases with hematopoietic malignances were detected by using RT-PCR, Western blot, and methylation-specific PCR. Of the 54 patients, 10 cases were low-risk MDS, 10 cases were high-risk MDS, 10 cases were acute myeloid leukemia (AML), 10 cases were acute lymphocytic leukemia (ALL), 10 cases were chronic myeloid leukemia in chronic phase (CML-CP), and 4 cases were CML in blast phase (CML-BP). 10 normal persons were studied as nective controls. The results showed that the incidence of p15INK4B methylation in cells of high-risk MDS was higher than that in low-risk MDS (6/10 VS 1/10, P=0.003), and the p15INK4B methylation was found to be associated with the down-regulation of the expressions of p15INK4B gene on both mRNA (r=−0.734, p<0.001) and protein (r=−0.664, p=0.001)levels, which indicated that the silencing of p15INK4B gene was in conjunction with hypermethylation in MDS. The expressions of p15INK4B on mRNA level and protein levels were almost detected in the MNCs from bone marrow of normal persons without the p15INK4B methylation. We also found the expression of DNMT3A and DNMT3B in high-risk MDS (densitometry readings respectively: 0.624±0.146, 0.577±0.344) were higher than in low-risk MDS (densitometry readings respectively: 0.487±0.300, 0.338±0.290) (P<0.05). The expression of DNMT1 was higher in the groups of low-risk MDS, high-risk MDS, AL and CML-CP( densitometry readings respectively: 0.487±0.218, 0.697±0.243, 0.706±0.463 and 0.867±0.375) than in normal control (densitometry reading: 0.181±0.312)(P<0.05, figure listed bellow), which indicated that up-regulated DNMTS might contribute to the hypermethylation of p15INK4B, and the higher expressions of de novo methyltransferases DNMT3A and DNMT3B may be related to the disease progression of MDS. The methylation of p15INK4B was also detected in 9/20 of AL cases accompanied by over-expressions of DNMT1, DNMT3A, and DNMT3B (densitometry readings respectively: 0.706±0.463, 1.066±0.547, and 0.530±0.428). The methylation of p15INK4B was detected in 1 of 10 cases of CML-CP patients, but all be detected in 4 case of CML-BP patients. These results indicated that the hypermethylation of p15INK4B gene may be one of the most common genetic event in pathogenesis of high-risk MDS, acute leukemia, and blast phase of CML. Furthermore, DNMT3A and DNMT3B were substantially over-expressed in the bone marrow cells of these patients. which might play an important role in the transformation from MDS to acute leukemia. Figure Figure

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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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Phase I clinical and laboratory evaluation of topotecan and cytarabine in patients with acute leukemia.

Journal of Clinical Oncology ◽

10.1200/jco.1997.15.1.44 ◽

1997 ◽

Vol 15 (1) ◽

pp. 44-51 ◽

Cited By ~ 49

Author(s):

K Seiter ◽

E J Feldman ◽

H D Halicka ◽

F Traganos ◽

Z Darzynkiewicz ◽

...

Keyword(s):

Bone Marrow ◽

High Risk ◽

Acute Leukemia ◽

S Phase ◽

Low Risk ◽

Myelogenous Leukemia ◽

Laboratory Evaluation ◽

Apoptotic Cells ◽

High Risk Patients ◽

Risk Patients

PURPOSE To determine the maximal-tolerated dose (MTD) of topotecan with cytarabine in acute leukemia patients, and to evaluate leukemia cell apoptosis in these patients. PATIENTS AND METHODS Fifty-three patients with acute leukemia not responsive to standard therapy were treated at eight dose levels of topotecan (2.5 mg/m2/d to 7.75 mg/m2/d). Topotecan was given as a 30-minute infusion daily with cytarabine 1 g/m2/d, both for 5 days. Using a flow-cytometric technique, the percent apoptotic cells in blood and bone marrow samples was determined, and the cell cycle distribution of the leukemic cells studied. RESULTS Oropharyngeal mucositis was dose-limiting. The MTD of topotecan was 4.75 mg/m2/d for 5 days in high-risk patients and 7.0 mg/m2/d for 5 days in low-risk patients. The mean percent apoptotic cells in the peripheral blood reached a peak of 18.8%, a median of 48 hours following the first dose of topotecan. Patients with higher S-phase fractions, either before treatment or following cytarabine, were more likely to achieve bone marrow aplasia than those with lower S-phase fractions (P = .01 and P < .05, respectively). Clinical responses were seen in four of 39 patients with acute myelogenous leukemia (AML; of whom 32 had received prior high-dose cytarabine), three of six with acute lymphoblastic leukemia (ALL), and one of eight with chronic myelogenous leukemia in blast phase (CML-BP). CONCLUSION The recommended phase II dose of topotecan with intermediate-dose cytarabine is 4.75 mg/m2/d for high-risk patients and 7.0 mg/m2/d for low-risk patients. The percentage of cells in S phase was important in determining response to treatment.

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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 ◽

10.1182/blood.v124.21.3179.3179 ◽

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.

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

Blood ◽

10.1182/blood.v128.22.5499.5499 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5499-5499 ◽

Cited By ~ 1

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.

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The Germline JAK2 GGCC (46/1) Haplotype and Survival Among 414 Molecularly-Annotated Patients with Primary Myelofibrosis

Blood ◽

10.1182/blood-2018-99-110046 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1761-1761

Author(s):

Ayalew Tefferi ◽

Terra L. Lasho ◽

Christy Finke ◽

Mythri Mudireddy ◽

Natasha Szuber ◽

...

Keyword(s):

Risk Factors ◽

High Risk ◽

Genetic Risk ◽

Triple Negative ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Version 2.0 ◽

Survival Effect

Abstract Background: We have long introduced the concept of host genetic variations in the phenotypic diversity of myeloproliferative neoplasms (MPN) (Blood 2008;111:2785). Previous studies have established an association between JAK2 mutations in myeloproliferative neoplasms (MPN) and the germline GGCC (46/1) haplotype, which constitutes a string of single nucleotide polymorphisms (SNPs) near the JAK2 gene that are inherited together on chromosome 9p (reviewed recently;Int J Mol Sci. 2018; 19: 1152). In 2010, we reported an association between shortened survival in primary myelofibrosis (PMF) and nullizygosity for the JAK2 46/1 haplotype (Leukemia 2010; 24:105), although our findings were not confirmed in another study (Leukemia 2010; 24:1533). Others have reported an association with splanchnic vein thrombosis, that was not accounted for by JAK2 mutations (Ann Hematol 2014;93:1845). In the current study, we have increased the number of informative cases to 414 (from 130 reported in 2010), in order to revisit with the phenotypic and prognostic relevance of the JAK2 46/1 haplotype in PMF. Methods : Study patients were recruited from the Mayo Clinic, Rochester, MN, USA. Diagnoses PMF and its leukemic transformation were confirmed by both clinical and bone marrow examinations, in line with the 2016 World Health Organization criteria (Blood. 2016;127:2391). Screening for the JAK2 46/1 haplotype included rs12343867 SNP genotyping, as previously detailed (Leukemia 2010; 24:105), and using a commercially available TaqMan SNP genotyping assay (Applied Biosystems Inc., Foster City, CA, USA). Statistical analyses considered clinical and laboratory data collected at the time of initial PMF diagnosis or Mayo Clinic referral point. Conventional statistics was used for confirming phenotypic associations and calculation of overall (OS) and leukemia-free (LFS) survival. The JMP® Pro 13.0.0 software from SAS Institute, Cary, NC, USA, was used for all calculations. Results: 414 patients with PMF (median age 63 years; 63% males) were included in the current study; among 324 evaluable cases, MIPSS70+ version 2.0 risk distribution was 18% very high risk, 41% high risk, 19% intermediate risk, 18% low risk and 4% very low risk. Driver mutation distribution was 63% JAK2, 17% type 1-like CALR, 3% type 2-like CALR, 7% MPL and 10% triple-negative. JAK2 46/1 haplotype was documented in 69% of the study patients, including 25% in homozygous and 44% in heterozygous state. Driver mutation frequency in patients homozygous/heterozygous/nullizygous for the 46/1 haplotype was 78%/60%/56% JAK2, 10%/20%/18% type 1-like CALR, 3%/2%/5% type 2-like CALR, 4%/8%/7% MPL and 6%/10%/14% triple-negative (p=0.02). The three 46/1 haplotype groups were phenotypically mostly similar, with the exception of platelet count (p=0.02) and leukocyte count (p=0.003), which were both higher with homozygous 46/1 haplotype. In univariate analysis, nullizygosity for the JAK2 46/1 haplotype was associated with inferior overall survival (HR 1.5, 95% CI 1.1-1.9; figure 1a); this survival effect was most pronounced in JAK2 mutated cases (figure 1b; p<0.001), as opposed to CALR/MPL mutated cases (figure 1c; p=0.48) or triple-negative cases (figure 1d; p=0.27). Multivariable analysis that included age and other genetic risk factors, including karyotype, driver mutational status and presence of high molecular risk mutations, such as ASXL1 and SRSF2, confirmed the independent prognostic contribution of nullizygosity for the 46/1 haplotype (p=0.02; HR 1.4, 95% CI 1.1-1.8). Nullizygosity for 46/1 also remained significant in the context of the recently unveiled genetics-based prognostic model, GIPSS (genetically-inspired prognostic scoring system) (p=0.04) (Leukemia.2018 doi: 10.1038/s41375-018-0107-z), but not in the context of MIPSS70+ version 2.0 (karyotype and mutation-enhanced international prognostic scoring system for transplant-age patients) (p=0.4). (JClinOncol.2018 doi: 10.1200/JCO.2018.78.9867). Leukemia-free survival was not affected by the 46/1 haplotype (p=0.6). Conclusions: The current study confirms the association of nullizygosity for the JAK2 GGCC (46/1) haplotype with inferior survival in PMF, primarily in JAK2-mutated cases; the observed survival effect was independent of currently acknowledged genetic risk factors, including karyotype and high molecular risk mutations. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2019-124575 ◽

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.

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