CALR-mutated Patients Research Articles

Effect of CALR and JAK2 mutations on the clinical and hematological phenotypes of the disease in patients with myelofibrosis - long-term experience from a single center.

Primary myelofibrosis (PMF) is a chronic clonal myeloid disorder. Together with essential thrombocythemia (ET) and polycythemia vera (PV), it belongs to a group of Philadelphia chromosome-negative myeloproliferative neoplasms. An integral part of laboratory tests carried out in this disease group is detecting the presence of mutations in the Janus kinase 2 gene at position 617 (JAK2 V617F) and in the gene encoding for the receptor for thrombopoietin (myeloproliferative leukemia virus oncogene, MPL) found in approximately 60% of PMF patients. The discovery of mutations affecting exon 9 of the calreticulin (CALR) gene was of great benefit to the diagnosis of the diseases in JAK2 V617F and MPL unmutated cases. This is a study of the effect of a mutation in the CALR gene on the clinical course in patients with primary, post-ET and post-PV myelofibrosis. Analysis of 66 patients (54.5% JAK2 V617F; 34.8% CALR; 6.1% MPL; 3.0% triple negative; 1.5% coincidence of CALR and JAK2 V617F) confirmed a different phenotype of the disease in CALR-mutated patients as compared with CALR-unmutated individuals. Those with CALR mutation were significantly younger and had borderline higher platelet counts, less pronounced splenomegaly and less frequent B symptoms at diagnosis. The study suggests that the driver mutation types define variations in the biological basis, clinical manifestations and course of the disease. The presence of CALR mutation has been shown to be an independent prognostic favorable factor. Careful risk stratification of these patients is of great importance to adequate therapeutic decision-making and aids in selecting high-risk patients eligible for allogeneic hematopoietic stem cell transplantation which continues to be the only treatment modality for myelofibrosis having curative potential.

CALR mutational status identifies different disease subtypes of essential thrombocythemia showing distinct expression profiles

Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia-negative myeloproliferative neoplasms (MPNs) characterized by erythrocytosis and thrombocytosis, respectively. Approximately 95% of PV and 50–70% of ET patients harbor the V617F mutation in the exon 14 of JAK2 gene, while about 20–30% of ET patients carry CALRins5 or CALRdel52 mutations. These ET CALR-mutated subjects show higher platelet count and lower thrombotic risk compared to JAK2-mutated patients. Here, we showed that CALR-mutated and JAK2V617F-positive CD34+ cells display different gene and miRNA expression profiles. Indeed, we highlighted several pathways differentially activated between JAK2V617F- and CALR-mutated progenitors, i.e., mTOR, MAPK/PI3K, and MYC pathways. Furthermore, we unveiled that the expression of several genes involved in DNA repair, chromatin remodeling, splicing, and chromatid cohesion are decreased in CALR-mutated cells. According to the low risk of thrombosis in CALR-mutated patients, we also found the downregulation of several genes involved in thrombin signaling and platelet activation. As a whole, these data support the model that CALR-mutated ET could be considered as a distinct disease entity from JAK2V617F-positive MPNs and may provide the molecular basis supporting the different clinical features of these patients.

Impact of molecular residual disease post allografting in myelofibrosis patients.

We screened 136 patients with myelofibrosis and a median age of 58 years who underwent allogeneic stem cell transplantation (AHSCT) for molecular residual disease for JAKV617F (n=101), thrombopoietin receptor gene (MPL) (n=4) or calreticulin (CALR) (n=31) mutation in peripheral blood on day +100 and +180 after AHSCT. After a median follow-up of 78 months, the 5-year estimated overall survival was 60% (95% confidence interval (CI): 50-70%) and the cumulative incidence of relapse at 5 years was 26% (95% CI: 18-34%) for the entire study population. The percentage of molecular clearance on day 100 was higher in CALR-mutated patients (92%) in comparison with MPL- (75%) and JAKV617F-mutated patients (67%). Patients with detectable mutation at day +100 or at day +180 had a significant higher risk of clinical relapse at 5 years than molecular-negative patients (62% vs 10%, P<0.001) and 70% vs 10%, P<0.001, respectively) irrespectively of the underlying mutation. In a multivariate analysis, high-risk diseases status (hazard ratio (HR) 2.5; 95% CI: 1.18-5.25, P=0.016) and detectable MRD at day 180 (HR 8.36, 95% CI: 2.76-25.30, P<0.001) were significant factors for a higher risk of relapse.

Impact of genotype on leukaemic transformation in polycythaemia vera and essential thrombocythaemia.

The influence of driver mutations on leukaemic transformation was analysed in 1747 patients with polycythaemia vera or essential thrombocythaemia. With a median follow-up of 7·2years, 349 patients died and 62 progressed to acute leukaemia or myelodysplastic syndrome. Taking death as a competing risk, CALR genotype was associated with a lower risk of transformation [subdistribution hazard ratio (SHR): 0·13, 95% confidence interval (CI): 0·2-0·9, P=0·039], whereas JAK2 V617F showed borderline significance for higher risk (SHR: 2·05, 95% CI: 0·9-4·6, P=0·09). Myelofibrotic transformation increased leukaemic risk, except in CALR-mutated patients. Next generation sequencing of 51 genes at the time of transformation showed additional mutations (median number: 3; range: 1-5) in 25 out of 29 (86%) assessable cases. Mutations (median: 1; range: 1-3) were detected in 67% of paired samples from the chronic phase. Leukaemia appeared in a JAK2 V617F negative clone in 17 (58%) cases, eleven of them being previously JAK2 V617F-positive. JAK2 V617F-mutated leukaemia was significantly associated with complex karyotype and acquisition of TP53 mutations, whereas EZH2 and RUNX1 mutations were more frequent in JAK2 V617F-negative leukaemia. Survival was longer in JAK2 V617F-unmutated leukaemia (343days vs. 95days, P=0·003). In conclusion, CALR genotype is associated with a lower risk of leukaemic transformation. Leukaemia arising in a JAK2 V617F-negative clone is TP53 independent and shows better survival.

CAL2 Monoclonal Antibody Is a Rapid and Sensitive Assay for the Detection of Calreticulin Mutations in Essential Thrombocythemia and May Provide Prognostic Informations

Background. Calreticulin (CALR) mutations are detected in the majority of JAK2 wild-type (wt) patients with Essential Thrombocythemia (ET) or Primary Myelofibrosis (MF). At variance with JAK2 and MPL point mutations, CALR mutations are highly heterogeneous, with several types of insdel reported to date, so that sequencing techniques are needed to cover the whole spectrum of mutations. CAL2 is a new monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR.Methods. We retrospectively analyzed 172 ET patients followed at our Institution from 1980 to 2015 who were tested for JAK2V617F at the time of diagnosis or during follow-up. In JAK2wt patients exon 9 CALR mutations were searched by PCR and capillary electrophoresis and MPLW515L/K by ARMS-PCR. In the same patients, bone marrow (BM) biopsies were immunostained with CAL2 and histologically reviewed for megakaryocytic features.Results. Median age at diagnosis was 54 years (range 14-87) and median time from first evidence of thrombocytosis to BM biopsy was 8.4 months (range 0-175). According to driver mutations, patients were classified as JAK2V617F-mutated (n=119, 69%), MPL-mutated (n=2, 1%), CALR-mutated (n=31, 18%) or triple-negative (n=20, 12%). As expected, CALR-mutated patients had significantly higher platelet count than JAK2V617F (799 vs 657 x 109/L, respectively; p=.019) and lower hemoglobin values (mean 13.6 vs 14.5 g/dL, respectively; p=.003). At a median follow-up of 9.8 years, incidence of thrombosis was significantly lower in CALR- than in JAK2V617F-mutated patients (3% vs 21%, respectively; p=.017), while no differences were observed in clinical presentation and long-term outcome between CALR-mutated and triple-negative patients. Concordance between molecular and immunohistochemical (IHC) detection of CALR mutations was optimal (CohenÕs kappa >0.8) but not complete, since 3 patients were positive by IHC only and 1 patient was positive by molecular only. In CAL2-positive BM samples (n=30) we defined 2 patterns (figure), characterized by staining of megakaryocytes only (pattern A, 37%) or staining of megakaryocytes and myeloid precursors (pattern B, 63%). Type B biopsies tended to have higher median cellularity (48% vs 30%; p=.22), higher median megakaryocytic number for HMF (20 vs 12.5; p=.14), higher frequency of megakaryocytic clusters (100% vs 60%; p=.014) and higher frequency of grade-1 fibrosis (81% vs 60%; p=.37) with respect to type A samples. One type A patient and 2 type B patients progressed to post-ET MF or acute leukemia. Moreover, 3 patients had a BM biopsy performed at the time of MF evolution but not at diagnosis of ET, and they displayed a type B pattern.Conclusions. CAL2 identifies CALR-mutated ET patients in a rapid, sensitive, specific and economic manner and it integrates molecular biology informations. Prognostic value of different IHC patterns should be confirmed in wider and indipendent series. [Display omitted] DisclosuresBonifacio:Ariad Pharmaceuticals: Consultancy; Amgen: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Research Funding.

Thrombotic risk correlates with mutational status in true essential thrombocythemia

True essential thrombocythemia (ET) may carry one of the known driver mutations (JAK2, MPL and CALR) or none of them [in triple-negative (3NEG) cases]. The patients' mutational status seems to delineate the clinical manifestations of ET. We report the data of 183 patients diagnosed with ET strictly according to the WHO 2008 criteria and with a full molecular diagnosis, including the following: 114 patients (62·3%) with JAK2V617F; 25 (13·7%) with CALR type 1 and 19 (10·4%) with CALR type 2; 3 (1·6%) with MPL; 22 (12%) who were 3NEG. Thrombotic risk was assessed by means of the IPSET-thrombosis score (IPSET-T). CALR and 3NEG patients had lower haemoglobin levels and leucocyte count than JAK2 patients. CALR patients, and those with type 2 in particular, had higher mean platelet counts and had extreme thrombocytosis more often than any of the other groups. Based on their IPSET-T stratification, 3NEG- and CALR-mutated patients belonged more frequently to the low-risk group and had a significant more favourable thrombosis-free survival rate than those with JAK2 mutation. These findings indicate that the three different molecular markers have a significant impact on the clinical course of true ET, giving rise to different phenotypes of the same disease.

Prognostic value of CALR vs. JAK2V617F mutations on splenomegaly, leukemic transformation, thrombosis, and overall survival in patients with primary fibrosis: a meta-analysis.

The understanding of genetic basis for Philadelphia-negative myeloproliferative neoplasm (MPN) has got much progress in recent years. But the effect of CALR vs. JAK2V617F mutations on the clinical progression and prognosis of primary fibrosis (PMF) remains relatively obscure. In this meta-analysis, we searched Pubmed, Embase, and Web of Science databases for observational studies published until February 2016. Researches that evaluated CALR vs. JAK2V617F mutations on PMF-relevant complications (splenomegaly, leukemic transformation, or thrombosis) and overall survival were selected. Pooled adjust odds ratio (OR), hazard risk (HR), and the corresponding 95% confidence intervals (CI) were calculated for the CALR-mutant versus the JAK2-mutant categories. Twelve studies involving 435 CALR-mutated and 1116 JAK2V617F PMF patients were analyzed. CALR-mutated patients displayed a lower risk of splenomegaly (OR 0.47, 95% CI 0.29-0.78) and thrombosis (OR 0.52, 95% CI 0.29-0.92) but showed no significant difference in the risk of leukemic transformation (OR 0.90, 95% CI 0.55-1.47) when compared with JAK2-mutated patients. CALR mutation favorably affected overall survival while JAK2 mutation led to poorer survival rate (HR 2.58, 95% CI 2.08-3.20). This meta-analysis confirmed that a genetic classification of PMF by CALR and JAK2 mutations carried significant prognostic relevance.

Disease characteristics and outcomes in younger adults with primary and secondary myelofibrosis.

Myelofibrosis (MF) is a rare haematopoietic disorder, commonly diagnosed in the 6th decade: less than 20% are diagnosed before the age of 50years. In this retrospective study we included all patients given a diagnosis of World Health Organization-defined primary or secondary MF when aged ≤50years. Forty-three patients with a median age of 43years were included. Median follow up was 44months. Twenty-two (51%) harboured the JAK2 V617F mutation, 18/43 (42%) CALR, 0/43 (0%) MPL mutations and 3/43 (7%) were 'Triple Negative' (TN). At the time of diagnosis, no significant differences existed in haematological and clinical phenotypes between JAK2, CALR and TN patients. The frequency of splenomegaly was greater (P=0·047) in the JAK2-mutated group compared to CALR-mutated patients. In the whole cohort, the 5-year probability of developing anaemia, thrombocytopenia and marked leucocytosis was 24%, 10% and 13% respectively. Finally, the cumulative incidence of thrombotic events and progression to acute myeloid leukaemia was 1% and 0·5% patient-year respectively. No death was reported during the follow-up. These findings suggest that MF in younger patients may have a more indolent course when compared to older patients.

Antiplatelet therapy versus observation in low-risk essential thrombocythemia with a CALR mutation.

The role of antiplatelet therapy as primary prophylaxis of thrombosis in low-risk essential thrombocythemia has not been studied in randomized clinical trials. We assessed the benefit/risk of low-dose aspirin in 433 patients with low-risk essential thrombocythemia (271 with a CALR mutation, 162 with a JAK2(V617F) mutation) who were on antiplatelet therapy or observation only. After a follow up of 2215 person-years free from cytoreduction, 25 thrombotic and 17 bleeding episodes were recorded. In CALR-mutated patients, antiplatelet therapy did not affect the risk of thrombosis but was associated with a higher incidence of bleeding (12.9 versus 1.8 episodes per 1000 patient-years, P=0.03). In JAK2(V617F)-mutated patients, low-dose aspirin was associated with a reduced incidence of venous thrombosis with no effect on the risk of bleeding. Coexistence of JAK2(V617F)-mutation and cardiovascular risk factors increased the risk of thrombosis, even after adjusting for treatment with low-dose aspirin (incidence rate ratio: 9.8; 95% confidence interval: 2.3-42.3; P=0.02). Time free from cytoreduction was significantly shorter in CALR-mutated patients with essential thrombocythemia than in JAK2(V617F)-mutated ones (median time 5 years and 9.8 years, respectively; P=0.0002) and cytoreduction was usually necessary to control extreme thrombocytosis. In conclusion, in patients with low-risk, CALR-mutated essential thrombocythemia, low-dose aspirin does not reduce the risk of thrombosis and may increase the risk of bleeding.

Impact of Minimal Residual Disease after Allografting Detected By JAK2V617F, MPL or Calreticulin in Myelofibrosis Patients

▪Introduction:Residual minimal disease (MRD) in hematological malignancies has become a valid tool to predict clinical relapse after allogeneic stem cell transplantation (ASCT).Methods and Patients:We screened 154 patients with myelofibrosis who underwent ASCT for molecular residual disease by qPCR or next-generation sequencing (NGS) for JAK2V617F, MPLW515L and MPLW515K or Calreticulin (L367fs*46, and K385fs*47) mutations in peripheral blood (PB) on days +100 and +180 after transplantation. Out of 154 pts, 103 were JAK2V617F (n=101), 31 Calreticulin (CALR), and 4 MPL mutated, while 13 pts were triple negative. 136 pts could be followed after ASCT with one molecular marker. The median age of the pts was 58 years (range, 32-75 y). Patients had either primary myelofibrosis (n= 90), post ET/PV myelofibrosis (n=40), myelofibrosis in acceleration or were transformed to AML (n=6). Conditioning mainly relied on a busulfan-based reduced-intensity regimen. Donor were HLA-identical sibling (n=26), matched unrelated (n=71) or mismatched unrelated (n=39). JAK2V617F, MPL, and CALR mutations were measured by usage of Taqman PCR or in case of CALR Type 2 mutation by digital PCR on day +100 and day +180 from PB as described elsewhere.Results:After a median follow up of 78 months (range, 49-101 months) the 5-year estimated overall survival was 60% (95% CI: 50-70%) and the cumulative incidence of relapse at 5 years was 26% (95% CI: 18-34%) for the entire study population. On days +100 and +180 after transplantation in 27% and 12% of the patients the underlying mutation was still detectable in peripheral blood. The percentage of complete clearance was higher in CALR-mutated patients (96%) in comparison to MPL (75%) and JAKV2617F (70%) mutated pts. Whereas there was a trend for better survival for CALR-mutated patients in comparison to JAK2-mutated patients (71% vs 57%; p=0.48), once a patient achieved molecular remission post-transplant the risk of relapse remained low independently of the underlying mutation. Patients who were alive and without relapse at days +100 or +180 but with still detectable mutation in PB had a significantly higher risk of relapse than those who were molecular negative (62% vs 10%, p<0.001; and 70% vs 10%, p<0.001, respectively). In a multivariate analysis only high-risk disease status (HR 2.5; 95% CI: 1.18-5.25, p=0.016) and detectable MRD at day 180 (HR 8.36, 95% CI: 2.76-25.30, p<0.001) were significant factors for a higher risk of relapse.Conclusions:JAK2V617F, CALR and MPL genetic lesions allow to monitor MRD in around 90% of the myelofibrosis patients after ASCT by qPCR or digital PCR in the PB. Persistence of MRD on days +100 or +180 in peripheral blood can be used to taper immunosuppressive drugs or to apply donor lymphocyte infusion to prevent clinical relapse. DisclosuresNo relevant conflicts of interest to declare.

Driver Mutations and Prognosis in 1118 Patients with Primary Myelofibrosis

▪Background: In primary myelofibrosis (PMF), ̴ 88% of patients harbor JAK2, CALR or MPL, with respective mutational frequencies of approximately 60%, 22% and 6%; ̴ 12% are wild type for all three driver mutations and are labelled as being "triple negative" (Blood. 2014;124:2507). It is now well-established that CALR-mutated patients with PMF display significantly better survival, compared to all the other mutational categories; in addition, preliminary data have suggested inferior survival for "triple-negative" cases (Leukemia. 2014;28:1472; Blood. 2014;124:1062) and the possibility that the favorable impact of CALR mutations might be restricted to CALR type 1 or type 1-like variants (Blood. 2014;124:2465). In the current study, we sought to clarify the prognostic impact of driver mutations, and CALR variants, on overall (OS) and leukemia-free (LFS) survival, in a two-center study involving 1118 patients.Methods: A total of 1118 patients with PMF from the Mayo Clinic (n =722) and University of Florence, Italy (n =396) were included in the current study. Study patients were selected based on availability of mutation information. PMF diagnosis was according to World Health Organization criteria (Blood. 2009;114:937). Previously 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 first referral for the Mayo cohort and date of diagnosis for the Florence cohort. Leukemic transformation events replaced death as the uncensored variable during LFS analysis and Cox proportional hazard regression model was used for multivariable analysis.Results:Analysis was first conducted on the Mayo cohort of 722 patients (median age 64 years; 64% males). DIPSS-plus risk distributions were 14% low, 17% intermediate-1, 37% intermediate-2 and 33% high. All patients were annotated for JAK2/CALR/MPL mutations as well as CALR variants; 477 harbored JAK2, 139 CALR and 41 MPL mutations; 65 patients were triple-negative. The 139 CALR -mutated patients included type 1/type 1-like (n =115) and type 2/type 2-like (n =24).At a median follow-up time of 3.1 years, 439 (61%) deaths and 63 (8.7%) blast transformations were documented. In univariate analysis, survival in patients with CALR type 1/type 1-like mutations was significantly longer than every other mutational category: HR (95% CI) were for triple-negative 2.7 (1.8-4.0), JAK2 2.7 (2.0-3.7), CALR type 2/type 2-like 2.3 (1.3-4.2) and MPL 1.9 (1.1-3.1) (Figure 1); these differences were sustained during multivariable analysis that included ASXL1 mutations (n =480) and DIPSS-plus, and were also apparent in the Florence cohort of 396 cases that included 251 JAK2, 53 CALR type 1/type 1-like, 21 CALR type 2/type 2-like, 50 triple-negative and 21 MPL mutated cases (Figure 2). There was no difference in survival among the Mayo cohort across mutational categories not including CALR type 1/type 1-like variants (p=0.33). Because specific driver mutations in PMF differentially cluster with age and sex, multivariable analysis including these two parameters was also performed and confirmed the significant survival advantage of CALR type 1/type 1-like over triple-negative, JAK2 and CALR type 2/type 2-like mutated cases.Based on the above information, we divided driver mutational categories into "type 1/type 1-like" (median survival 10.3 years) and "all other mutational categories" (median survival 3.9 years; p<0.01). The difference in survival between these two groups remained significant (HR 2.1, 95% CI 1.5-2.9) during multivariable analysis that included both DIPSS-plus (P<0.01) and ASXL1 mutation (HR 1.8, 95% CI 1.4-2.2). LFS was similar between these two groups (p=0.4) but it was inferior in triple-negative cases compared to CALR type 1/type 1-like variants (HR 2.8, 95% CI 1.3-6.2), MPL (HR 4.6; 95% CI 1.04-20.4) and JAK2 (HR 2.5, 95% CI 1.3-4.8) but not CALR type 2/type 2-like variants (p=0.56). There was no difference in LFS among mutational categories not including triple-negative cases (p=0.33).Conclusions: In PMF, driver mutations might be classified into two prognostically distinct categories: "favorable" (type 1/type 1-like) and "unfavorable" (all other mutational categories). In addition, triple-negative cases might be at a higher risk of leukemic transformation. [Display omitted] [Display omitted] DisclosuresPardanani:Stemline: Research Funding. Vannucchi:Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Driver Mutations and Prognosis in 502 Patients with Essential Thrombocythemia

▪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. [Display omitted] [Display omitted] DisclosuresPardanani:Stemline: Research Funding.

New and treatment-relevant risk stratification for thrombosis in essential thrombocythemia and polycythemia vera.

New and treatment-relevant risk stratification for thrombosis in essential thrombocythemia and polycythemia vera.

Mutations and long-term outcome of 217 young patients with essential thrombocythemia or early primary myelofibrosis.

We investigated the influence of molecular status on disease characteristics and clinical outcome in young patients (⩽ 40 years) with World Health Organization (WHO)-defined essential thrombocythemia (ET) or early/prefibrotic primary myelofibrosis (early-PMF). Overall, 217 patients with ET (number 197) and early-PMF (number 20) were included in the analysis. Median follow-up time was 10.2 years. The cumulative incidence of thrombosis, hemorrhages and disease evolution into myelofibrosis/acute leukemia were 16.6%, 8.6% and 3% at 15 years, respectively. No differences were detectable between ET and early-PMF patients, although the latter cohort showed a trend for worse combined-event free survival (EFS). Mutation frequency were 61% for JAK2V617F, 25% for CALR and 1% for MPLW515K, and were comparable across WHO diagnosis; however, JAK2V617F allele burden was higher in the early-PMF group. Compared with JAK2V617F-positive patients, CALR-mutated patients displayed higher platelet count and lower hemoglobin level. CALR mutations significantly correlated with lower thrombotic risk (9.1% versus 21.7%, P = 0.04), longer survival (100% versus 96%, P = 0.05) and better combined-EFS (86% versus 71%, P = 0.02). However, non-type 1/type 2 CALR mutations ('minor' mutations) and abnormal karyotype were found to correlate with increased risk of disease evolution. At last contact, six patients had died; in five cases, the causes of death were related to the hematological disease and occurred at a median age of 64 years (range: 53-68 years). Twenty-eight patients (13%) were unmutated for JAK2, CALR and MPL: no event was registered in these 'triple-negative' patients.

Primary Trombocythemia in Children and Adolescents Includes Different Subtypes Compared to Adult Essential Thrombocythemia

The recent discovery of various mutations of the CALR gene that are mutually exclusive with JAK2 and MPL mutations has allowed a correct diagnosis in about 90% of adult cases of essential thrombocythemia (ET). Moreover, the mutation status of JAK2 and CARL defines subtypes of ET in adults with a substantially different clinical course and outcome. Based on our experience, we suggested that primary thrombocythemia (PT) in children is characterized by subtypes that differ from those found in adult ET. The present study was carried out in children and adolescents with PT in order to (a) characterize the various subtypes of the disease and (b) analyze their clinical and biologic features, treatment approach and outcome.PT patients aged <20 years (yrs) at diagnosis (dx) were evaluated for mutations of JAK2, thrombopoietin (TPO) and its receptor (MPL) and CALR genes, and for clonal hematopoiesis (females). The presence of MPLS505A (confirmed on DNA from buccal swabs) defined a hereditary thrombocytosis (HT). ET was diagnosed according to WHO 2008 criteria. For wild type patients, an additional inclusion criteria was a follow-up >24 months.Among 58 PT patients (males: 23; females: 35; median age at dx: 14.4 yrs), 21 (36%) had HT due to MPLS505A, 14 were JAK2V617F-mutated (24%), 9 (16%) harbored CALR mutations and 14 (24%) were wild type for JAK2, CALR and MPL (Fig 1).JAK2- and CALR-mutated were older than those with wild type ET or with HT (median age, 17.6 and 16.1 vs 10.4 and 13.7 yrs, p .028). As to the hematologic findings, HT patients showed both hematocrit values (median, 36.3%) and leukocytes counts (median, 9.53 x109/L) significantly lower than ET patients, whatever the subtypes (median, 41.2% and 11.2 x109/L, p .006 and p .029, respectively). No differences were found with regard to platelets both between HT and ET and among the different ET subtypes. JAK2-mutated patients exhibited more frequently symptoms (69%) compared to CALR-mutated (22%), wild-type ET (14%) and HT (14%) patients (p. 0057). Splenomegaly at diagnosis was recorded more frequently in JAK2-mutated than in CALR-mutated or wild type-ET or HT (50%, 33% 21% and 14% , respectively, p .122).Antiplatelet agents, mostly acetylsalicylic acid (ASA), were started less frequently in HT than in ET patients, irrespective of the subtypes (57% vs 81%, p .05). The use of ASA progressively decreased over the time; at the last follow-up, 2 patients with HT, 2 CALR-mutated and 1 JAK2-mutated patients were still receiving ASA, while no wild type ET patient was on treatment. Cytoreductive agents, hydroxyurea and/or interferon and/or anagrelide, were used in a minority of HT patients (19%) in comparison with ET patients (65%), p .001, mainly with those wild-type (78%, p <.001). At the last observation, one HT patient was still receiving cytoreductive agents compared to 30% of ET patients whatever the subtypes (p .024). After a median follow-up of 196 months (similar in the different subtypes), all patients are alive. On the whole, 5 thrombotic events were recorded in 3 patients with HT and in 2 ET patients (1 JAK2-mutated and 1 JAK2 and CALR wild-type), without any significant thrombophilic abnormalities during treatment with ASA and/or cytoreductive agents. A progressive splenomegaly was recorded in 9 (15%) patients (2 HT, 4 JAK2-mutated, 3 CALR-mutated) and it was combined with grade ≥2 medullar fibrosis in 2/4 JAK2-mutated and in 2/3 CALR-mutated patients. None of the JAK2 and CALR wild-type patients had spleen enlargement or reticulin fibrosis (p .022). Two untreated patients (1 HT and JAK2 and CALR wild-type) developed malignancies.On the whole, these data emphasize that in young patients with PT, hereditary forms can be frequently observed. Thrombotic events, recorded mainly in HT patients despite treatment with ASA, were probably due to a MRP4 protein overexpression that was found in our MPLS505A HT. Moreover, our observations highlight that, in contrast to adult ET, more than one third of young ET patients have no JAK2 or CALR mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MRP4 Expression and Platelet Activation in Children and Adolescents with Different Subtypes of Primary Thrombocythemia

Background and aims. Essential thrombocythemia (ET) rarely occurs in children and adolescents. In our experience, 40% of pediatric patients with primary thrombocythemia (PT) have JAK2 V617F or CALR mutations, 24% have a diagnosis of ET without any known molecular markers while 36% have hereditary thrombocytosis (HT) with a MPLS505A mutation. Thrombotic events, frequent in adult ET presenting high-risk factors and rare in pediatric PT, have been observed in pediatric HT patients with MPLS505A mutation in treatment with aspirin (ASA). The multidrug resistance protein-4 (MRP4) is an ATP-binding cassette transporter involved in the efflux of several pharmacological and physiological compounds. MRP4 has been identified as a modulator of ASA action in platelets; in addition, it also influences platelet activation. Recent studies have shown that MRP4 over-expression has a role in reducing the effect of ASA in patients who have undergone a bypass surgical procedure and that ASA induces platelet MRP4 upregulation. Aims of this study were to evaluate and correlate MRP4 expression and platelet function in children and adolescents aged <20 years at diagnosis with different subtypes of PT.Methods. MRP4 protein and mRNA expressions were evaluated in platelets obtained from healthy volunteers (HV) and from 41 PT patients: M/F ratio: 0.78; median age at diagnosis: 14.5 years; median platelet count at study: 671x109/L. Ten patients had MPLS505A mutation (HT), 10 were JAK2V617F-mutated (ET) and 6 harbored CALR mutations (ET) while 15 cases were JAK2, CALR and MPL wilde-type (ET). Expression of MRP4 protein and mRNA were analyzed by Western blot and RT-PCR, respectively, and the results were reported as ET/HV or HT/HV expression values. Platelet aggregation, using ADP at different concentration (0.8 to 2 μM) and collagen (1 μg/ml) as agonists, and platelet secretion, expressed as ATP release after U46619 + epinephrine (1 + 20 μM), using the luciferin-luciferase assay, were utilized as platelet function tests. This study was conducted in accordance with the Declaration of Helsinki.Results. Protein MRP4 expression was higher both in HT (4.23+/-1.88) and in ET patients with CALR mutations (4.27+/-2.60) compared to the values found in wild-type ET patients (3.55+/-1.52). In JAK2V617F-mutated ET patients, the MRP4 protein expression (2.03+/-1.46) was significantly lower compared to the values observed in all other ET and HT patients (p .05), whereas the MRP4 mRNA expression was significantly higher (ΔΔCt 0.021+0.003) compared both to HT patients (0.009+/-0.0039 ΔΔCt) (p .01) and CALR-mutated patients (ΔΔCt 0.014+/- 0.006). Patients with HT showed a significantly higher response to ADP 0.8 µM (83+/-29 Mx%) compared to all subgroups of ET patients who showed a similar response (60+/-34 Mx%; p .01). A significantly shorter lag-phase in response to collagen (1 µg/ml) was observed in HT compared to ET patients (33+/-3 sec vs 51+/-4 sec, p .005). Among the ET population, JAK2-mutated patients showed a significantly shorter lag-phase in response to collagen compared to wild-type patients (39+/-12.5 sec vs 50+/-15 sec), p .011. Platelet secretion was significantly higher in HT compared to ET patients, p .001.Conclusions. This study for the first time provides evidence that children and adolescents with MPLS505A-mutated HT show a higher platelet reactivity compared to age-matched patients with ET. Moreover, platelet reactivity correlates with MRP4 protein overexpression. These findings help to shed light into the thrombotic events observed in HT MPLS505A patients despite treatment with ASA. DisclosuresNo relevant conflicts of interest to declare.

Characterization of CD34+ Hematopoietic Progenitor Cells in JAK2V617F and Calr-mutated Myeloproliferative Neoplasms

Introduction: The dominance of the JAK2V617F-positive clone at the CD34+ compartment is an important modifier of the disease phenotype in myeloproliferative neoplasms (MPN). Recently, mutations in the calreticulin gene (CALR) have been described in around 40-70% of JAK2V617F and MPL wild-type essential thrombocythemia (ET) and myelofibrosis (MF) patients. However, there is limited information regarding the role of CALR mutant clone in hematopoietic progenitor cells.Objective: To study the mutant allele burden at progenitor level in JAK2V617F-positive and CALR-mutated MPN.Methods: Sixty-five patients with MPN including 36 with polycythemia vera (PV) all JAK2V617F-positive, 13 with ET (7 JAK2V617F-positive and 6 CALR-mutated) and 16 with MF (9 JAK2V617F-positive post-PV MF, 4 CALR-mutated primary MF and 3 CALR-mutated post-ET MF) were included in the study. Granulocytes were isolated from peripheral blood by density gradient, whereas CD34+ cells were purified by immunomagnetic positive selection. Stem cells (CD34+CD38-) and progenitors (CD34+CD38+) populations were further separated by fluorescence-activated cell sorting. JAK2V617F and CALR allele burden was measured by quantitative PCR and PCR followed by fragment analysis, respectively, in stem cells, progenitor cells and granulocytes. The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki.Results:CALR-mutated ET patients harbored a higher mutant load in CD34+CD38- than JAK2V617F-positive ET patients (30.6 vs 6.3%, p=0.01), whereas no significant differences were observed in CD34+CD38+ and in granulocytes allele burdens. Moreover, CALR-mutated ET patients showed a higher mutational load in CD34+CD38- than JAK2V617F-positive PV (30.6% vs 15.7%, p=0.04) but the mutant load in granulocytes was lower (29.6% vs 63.3%, p<0.001).The mutant allele burden in granulocytes and CD34+ cells was higher in patients with JAK2V617F-positive MF than in those with CALR-mutated MF (CD34+CD38-: 71% vs 47.2% p=0.05, CD34+CD38+: 68.4% vs 40.6% p=0.018, granulocytes: 76.9% vs 53.7% p=0.05).Finally, we could demonstrate that the mutant load was lower in CALR-mutated ET patients than in CALR-mutated MF at progenitor level and in granulocytes (CD34+CD38-: 30.6% vs 47.1% p=0.08, CD34+CD38+: 17.8% vs 40.6% p=0.03, granulocytes: 29.6% vs 53.7% p=0.004).Conclusion:CALR-mutated ET patients have a higher mutant load in CD34+CD38- than JAK2V617F-positive ET and PV patients, whereas the JAK2V617F-positive hematopoietic progenitor cells have more differentiation potential than those CALR-mutated. Moreover, in the MF phase of MPN, the expansion of the mutated clone at the progenitor level is greater in JAK2V617F-positive than in CALR-mutated patients. DisclosuresNo relevant conflicts of interest to declare.

Monitoring of Calr Allele Burden in Patients with Essential Thrombocythemia Treated with Imetelstat, a Telomerase Inhibitor, Reveals Rapid and Substantial Molecular Responses

Background: In essential thrombocythemia (ET), mutations in the calreticulin gene (CALR) are found in the majority of patients that are negative for mutations in the JAK2 and MPL genes. Patients with mutated CALR have a better prognosis and lower thrombosis risk than those with mutated JAK2. Recently, decreases in the CALR mutant allele burden have been observed with interferon alpha after long-term treatment of two- and four-years, respectively (NEJM 2014, 371;2:188-9, Cassinat B. et al.). From the clinical phase II ET study with imetelstat (IT), a first in class, potent, specific inhibitor of telomerase, we reported a substantial and rapid decrease in the JAK2V617F allele burden. 7/8 patients (88%) reached a partial molecular response (MR: >50% reduction from baseline), 6 within the first 3 months and 1 after 12 months.Aims: We aimed to monitor molecular response to imetelstat therapy in ET patients with CALR mutations by serial measurements of CALR mutant allele burden.Methods: The study enrolled patients with ET who had failed or were intolerant to ≥1 prior therapy, or refused standard therapy. During the induction phase, patients were treated with IT 7.5 mg/kg or 9.4 mg/kg IV weekly until attainment of platelet (plt) count ~250-300x109/L. Maintenance phase was then commenced with dosing titrated to platelet count. CALR mutations were detected by Sanger sequencing and quantification of the allele burden was performed by fragment analysis.Results: 18 patients with ET (10 patients with JAK2V617F, 5 patients with CALR and 2 patients with MPL mutations) were enrolled and were treated in the study. 4 of the 5 CALR positive patients achieved complete hematologic responses (CR: Plts < 400 x109/L for 4 weeks) after a median of 6 weeks (range 5 to 14 weeks) and the 5th patient achieved a partial response after 19 weeks, with weekly imetelstat doses starting at 7.5 mg/kg in 2 patients and 9.4 mg/kg in 3 patients. CALR mutations consisted of three cases with type 1 (52-bp deletion; c.1092_1143del), one with type 2 (5-bp insertion; c.1154_1155insTTGTC) and one unknown mutation type (32-bp deletion; c.1092_1124del). Molecular monitoring of CALR allele burden at cycles 3, 6 and 9 demonstrated a rapid decrease in the CALR-mutated patients. 3 pts had a 35-50% reduction from baseline within 4 months and 1 pt had an 11% decrease within 8 months. One of these patients had a 48% reduction in 2 months and a second one had a deepening of response after 10 months to a 55% reduction. All 3 patients with CALR allele burden reductions of 35% or more also achieved hematologic CR.Conclusions: In 4 of 5 patients with CALR-mutated ET, IT induced a rapid CR and in 3 patients hematologic CR was associated with a substantial decrease in the allele burden of 35-50% after 4 months which is more rapid than what has so far been seen with other treatments for ET. Overall 9/13 patients with JAK2 or CALR mutations reached a >35-50% decrease of the mutant clone within 4 months of treatment with IT, providing clinical confirmation of imetelstat’s inhibition of neoplastic clonogenic cell growth in vivo. This additional evidence of reduction in the clonal burden supports IT’s potential to modify the biology of MPNs long-term. DisclosuresBaerlocher:Geron Corporation: Research Funding. Odenike:Incyte Pharmaceuticals, Sanofi Aventis, Suneisis, Algeta, Spectrum Pharaceuticals: Honoraria. Roeth:Geron Corporation: Research Funding. Shih:Geron: Employment, Equity Ownership. Burington:Geron Corporation: Employment, Equity Ownership. Leibundgut:Geron Corporation: Research Funding.

Calreticulin Gene Mutations in Suspicion of Myeloproliferative Neoplasms: A Single Institution Experience

Recently, Klampfl et al. and Nangalia et al. reported recurrent somatic mutations in the calreticulin (CALR) gene in patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 and MPL. All CALR mutations described to date are either deletions or insertions, and occur in exon 9 and result in a frameshift leading to a loss of KDEL sequence (endoplasmic reticulum retention motif) and multiple calcium biding sites with a new basic (instead of acidic) C terminal region.The aim of this work is to know how far the CALR mutations can fill the molecular diagnostic gap for myeloproliferative neoplasms (MPNs) left by JAK2 and MPL mutations.The study was approved by the local ethics committee.Our study population consists of 155 patients with platelet counts > 500 G/L sampled between 2012 and 2013 for suspicion of MPNs. Only JAK2 V617F negative patients were included.Analyses were performed using genomic DNA isolated from peripheral blood. Patients were screened for CALR mutations using a High Resolution Melting (HRM) and then the precise mutations were characterized by Sanger sequencing which is a new approach for this test.A total of 21 (13.5%) patients with CALR mutations were detected with 8 distinct variants.Among CALR mutations, type 1 (52 basepair deletion, c.1092_1143del) and type 2 (5 basepair insertion, c.1154_1155insTTGTC) were the most common (respectively 7 (33%) and 7 (33%) cases). Three patients had 2 concurrent mutations (insertion+substitution / Deletion+substitution / deletion +insertion). We found a deletion that has not been reported so far to our knowledge (c.1125_1147del). Sequencing is ongoing for 4 patients.Compared with negative patients, CALR-mutated patients demonstrated markedly higher platelet counts (median count: 820 vs 543 G/L). DisclosuresNo relevant conflicts of interest to declare.

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

▪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. DisclosuresNo relevant conflicts of interest to declare.