Molecular Correlates of Anemia in Primary Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4068-4068
Author(s):  
Daniela Barraco ◽  
Terra L. Lasho ◽  
Kebede H. Begna ◽  
Naseema Gangat ◽  
Christy Finke ◽  
...  
Keyword(s):  
Triple Negative ◽  
Primary Myelofibrosis ◽  
Hemoglobin Level ◽  
Older Age ◽  
Driver Mutation ◽  
World Health ◽  
Driver Mutations ◽  
Severe Anemia

Abstract Background : Anemia is one of the most prominent symptoms in primary myelofibrosis (PMF) and is often associated with inferior quality of life and survival. Current drugs, including JAK inhibitors, are suboptimal in the treatment of PMF-associated anemia and better information on its pathogenesis is critical for the development of more effective drugs. In the current study of JAK2/CALR/MPL annotated patients with PMF, we examined its correlation with both "driver" and "non-driver mutations", as well as cytogenetic abnormalities, in order to gain better insight into its pathogenesis. Methods: Study patients were selected based on availability of "driver" 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). Considering their relatively high mutational frequency in PMF, subsets of patients were also screened for ASXL1, spliceosome component (SF3B1, U2AF1, SRSF2, ZRSR2) and TET2 mutations. Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature. Statistical analyses considered clinical and laboratory parameters obtained at time of first referral at the Mayo Clinic. Results : Analysis was conducted on 722 patients (median age 64 years; 64% males). DIPSS-plus risk distribution was 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). Non-driver mutations screened included ASXL1 (n =480; mutated 38%), SRSF2 (n =474; mutated 14%), U2AF1 (n =457; mutated 16%), SF3B1 (n =328; mutated 8%), ZRSR2 (n =180; mutated 11%) and TET2 (n =180; mutated 18%). Karyotype was normal in 60%, favorable in 28% and unfavorable in 12%. Anemia was defined as being absent (normal sex-adjusted hemoglobin level; n =110; 15%), mild (hemoglobin level of ≥10 g/dL but below sex-adjusted normal value; n =263; 36%), moderate (hemoglobin level below 10 g/dL but not transfusion-dependent; n =108; 15%) and severe (transfusion-dependent anemia; n =241; 33%). Presence of at least mild anemia was associated with abnormal karyotype (p=0.006) with no difference between favorable and unfavorable abnormalities, U2AF1 (p=0.002), TET2 (p=0.02) and ASXL1 (p=0.04) mutations; other significant associations included male sex and older age. Presence of moderate to severe anemia was associated with U2AF1 (p<0.0001), SRSF2 (p=0.007) and driver mutations other than CALR type 1/type 1-like (p<0.0001). Presence of severe anemia was associated with U2AF1 (p<0.0001), SRSF2 (p=0.003) and non-CALR driver mutations (17% incidence in both types of CALR variants vs 51% in triple negative, 35% JAK2, 39% MPL mutated cases; p<0.0001). An association with older age but not gender was also noted for both moderate to severe and severe anemia (p<0.0001). During multivariable analysis, independent associations with moderate to severe anemia were confirmed for U2AF1 (p<0.0001), SRSF2 (p=0.007) and age (p=0.0001), but not driver mutation profile (p=0.30). A similar analysis for severe anemia also identified U2AF1, SRSF2 and age as being significantly relevant. Conclusions : The current study identifies older age and the spliceosomal mutations U2AF1 and SRSF2 as having strong and independent association with moderate to severe anemia in PMF. Targeting the spliceosome machinery or its mutant components offers a potential approach in the treatment of PMF-associated anemia. Disclosures Pardanani: Stemline: Research Funding.

scholarly journals U2AF1 Mutation Variants and Their Phenotypic and Prognostic Relevance in Primary Myelofibrosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4248-4248 ◽  
Author(s):  
Ayalew Tefferi ◽  
Daniela Barraco ◽  
Terra L. Lasho ◽  
Christy Finke ◽  
Sahrish Shah ◽  
...  
Keyword(s):  
Univariate Analysis ◽  
Multivariable Analysis ◽  
Normal Karyotype ◽  
Primary Myelofibrosis ◽  
Older Age ◽  
Severe Anemia ◽  
Mutational Status ◽  
Survival Effect

Abstract Background U2AF1 mutations occur in approximately 16% of patients with primary myelofibrosis (PMF) and were significantly associated with anemia, thrombocytopenia, older age, JAK2V617F, ASXL1 mutations and normal karyotype (Leukemia 2014;28:431); furthermore, U2AF1 mutations were associated with inferior survival in univariate but not multivariable analysis. In the current study, we looked for the possibility of a differential effect from U2AF1 mutation variants on these observations in PMF. Methods Study patients fulfilled the 2016 WHO criteria for the diagnosis of PMF (Blood. 2016;127:2391). Additional selection criteria included the availability of U2AF1 mutational status. Previously published methods (Leukemia 2014;28:431), including targeted next generation sequencing (Blood 2015 126:354), were used to screen for U2AF1 and other prognostically-relevant mutations. Statistical analyses considered clinical and laboratory parameters obtained at time of initial referral to the Mayo Clinic. Results Patient characteristics: U2AF1 mutational status was available for 457 patients and 72 (16%) harbored one of several mutation variants: these mutations affected residue Q157 in 44 (10%) patients, S34 in 24 (5%) and the remaining 1% displayed other variants. The 44 patients with U2AF1Q157 mutations included 23 with Q157P, 18 with Q157R and 3 with Q157-Y158insYE. The 24 patients with U2AF1S34 included 16 with S34F and 8 with S34Y. Only one patient harbored both Q157 and S34 mutations (Q157R, S34Y). Among all 457 study patients, median age was 63 years, 64% were males, dynamic international prognostic scoring system (DIPSS)-plus (JCO 2011;29:392) risk distributions were 13% low, 18% intermediate-1, 38% intermediate-2 and 32% high and driver mutation distributions were 54% JAK2, 22% CALR type 1/ type1-like, 4% CALR type 2/type 2-like, 7% MPL and 13% triple-negative. Cytogenetic studies were available in 449 patients: 39% abnormal and 10% unfavorable. All 457 patients were screened for ASXL1 mutations with 37% mutated, 450 for SRSF2 mutations with 15% mutated, 403 for SF3B1 with 8% mutated, 366 for IDH1/2 with 5% mutated and 369 for EZH2 with 4% mutated. Median follow-up was 4.4 years and during this period, 318 (70%) deaths and 53 (12%) leukemic transformations were documented. Phenotypic correlates of U2AF1 mutation variants: Because of the relatively small number of informative cases with specific mutations, we classified all patients into Q157 (n=44) and S34 (n=24) mutation variants and compared them with the 385 U2AF1 un-mutated cases. First, we confirmed our previous observations regarding the association between U2AF1 mutations in general and older age (p=0.0003), JAK2V617F (p<0.0001), ASXL1 mutations (p=0.0002), normal karyotype (p=0.03), hemoglobin <10 g/dL (p<0.0001) and platelet count <100 x 10(9)/L (p<0.0001); when the two U2AF1 mutation categories were analyzed separately, the corresponding p values for Q157 were 0.0005, 0.001, <0.0001, 0.04, <0.0001 and <0.0001 and for S34 were 0.12, 0.001, 0.41, 0.41, <0.0001 and 0.67. Phenotypic correlates of U2AF1 mutation variants: In univariate analysis, survival was adversely affected by U2AF1Q157 (p<0.0001; HR 2.2, 95% CI 1.6-3.1) but not by U2AF1S34 (p=0.8; HR 1.1, 95% CI 0.6-1.8) mutations (Figure 1a). Furthermore, the negative survival effect of U2AF1Q157 mutations was independent of anemia, thrombocytopenia, ASXL1, SRSF2, IDH1/2 and EZH2 mutations, as well as driver mutational status; multivariable analysis that included all molecular alterations identified U2AF1Q157 (HR 1.6, 95% CI 1.1-2.3), ASXL1 (HR 2.3, 95% CI 1.8-3.5), SRSF2 (HR 1.6, 95% CI 1.2-2.2) and absence of CALR type-1/like (HR 2.6, 95% CI 1.8-3.5) mutations as independent risk factors for survival. Finally, the survival effect of U2AF1Q157 mutations was independent of DIPSS-plus in patients with hemoglobin ≥10 g/dL (HR 2.6, 95% CI 1.3-5.3; p=0.007) (Figure 1c) but not in those with hemoglobin <10 g/dL (p=0.98) (Figure 1b). Conclusion Both U2AF1Q157 and U2AF1S34 mutation variants in PMF are associated with JAK2V617F and severe anemia whereas only the former is associated with ASXL1 mutations and thrombocytopenia. More importantly, U2AF1Q157, but not U2AF1S34, mutation variants in PMF are predictive of inferior survival, independent of other adverse mutations, and, in the absence of severe anemia, independent of DIPSS-plus. Disclosures No relevant conflicts of interest to declare.

Driver Mutations and Prognosis in 1118 Patients with Primary Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2801-2801 ◽  
Author(s):  
Ayalew Tefferi ◽  
Paola Guglielmelli ◽  
Terra L. Lasho ◽  
Yoseph Elala ◽  
Tiziana Fanelli ◽  
...  
Keyword(s):  
Research Funding ◽  
Triple Negative ◽  
Multivariable Analysis ◽  
Primary Myelofibrosis ◽  
Driver Mutations ◽  
Leukemic Transformation ◽  
Advisory Committees ◽  
University Of Florence

Abstract 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. Disclosures Pardanani: 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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1599-1599 ◽  
Author(s):  
Yoseph Elala ◽  
Terra L. Lasho ◽  
Naseema Gangat ◽  
Christy Finke ◽  
A Kamel Abou Hussein ◽  
...  
Keyword(s):  
Platelet Count ◽  
Survival Data ◽  
Triple Negative ◽  
Univariate Analysis ◽  
Multivariable Analysis ◽  
Driver Mutations ◽  
Mutational Status ◽  
Age And Sex

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

The clinical and prognostic relevance of driver mutations in 203 Taiwanese patients with primary myelofibrosis

2017 ◽  
Vol 71 (6) ◽  
pp. 514-521 ◽  
Author(s):  
Ming-Chung Kuo ◽  
Tung-Huei Lin ◽  
Chien-Feng Sun ◽  
Tung-Liang Lin ◽  
Jin-Hou Wu ◽  
...  
Keyword(s):  
Triple Negative ◽  
Direct Sequencing ◽  
Primary Myelofibrosis ◽  
Driver Mutations ◽  
Driver Genes ◽  
Allele Burden ◽  
Prognostic Relevance ◽  
Mutational Status ◽  
Length Changes

AimsWe investigated the clinical and prognostic relevance of the mutational status of driver genes with allele burden and endogenous erythroid colony (EEC) growth in 203 Taiwanese patients with primary myelofibrosis (PMF).MethodsPyrosequencing was used to detect JAK2V617F mutational status and measure allele burden, while MPL (exon 10) mutations were analysed by PCR assay and then by direct sequencing. CALR exon 9 mutations were first screened for length changes by GeneScan followed by sequencing. The allele burden of the mutated CALR gene was measured by pyrosequencing. The EEC assay was conducted using a serum-free culture system.ResultsThe frequencies of the three driver mutations and triple-negative status were similarly distributed between pre-PMF and overt PMF patients, except that pre-PMF patients had a higher incidence of CALR type 2/type-2 like mutations and a lower JAK2V617F allele burden. EEC growth and CALR mutations conferred favourable overall survival (OS). A lower JAK2V617F allele burden and grade 3 bone marrow fibrosis were associated with shorter OS and decreased leukaemia-free survival (LFS). Type 2/type 2-like CAL mutations were associated with better LFS compared with type1/type 1-like mutations. Patients with triple-negative mutation status had significantly worse OS and LFS. The allele burden of CALR mutations remained unchanged, while some JAK2V617F mutations showed clonal expansion in patients during secondary acute myeloid leukaemia transformation.ConclusionsOur study showed that EEC growth, a higher JAK2V617F allele burden and CALR mutations, especially type 2, were independent predictors for better outcomes in PMF. The allele burden of CALR mutations remained stable, but the allele burden of JAK2V617Fmutations was variable during leukaemia transformation.

A Greater Mutational Complexity May Contribute to the Differential Prognostic Impact of Type 1/Type 1-like Versus Type 2/Type2-like Calreticulin Mutations in Primary Myelofibrosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1627-1627
Author(s):  
Paola Guglielmelli ◽  
Giada Rotunno ◽  
Tiziana Fanelli ◽  
Annalisa Pacilli ◽  
Laura Calabresi ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Chronic Phase ◽  
Jak2 V617f ◽  
Primary Myelofibrosis ◽  
Driver Mutations ◽  
Prognostic Impact ◽  
Advisory Committees ◽  
Significant Difference

Abstract BACKGROUND. Calreticulin (CALR) mutations (mut) are associated with a IPSS/DIPSSplus and mutated ASXL1-independent favorable outcome in patients (pts) with primary myelofibrosis (PMF) compared to those harboring JAK2 V617F and MPL W515 mut and those lacking driver mutations ("triple negative", TN). Spliceosome mut were reported to be less represented in CALR - mut pts compared with other genotypes (Leukemia 2014;28:1472), but no other molecular or cytogenetic correlate is known. It is also debated whether there are prognostic differences between type 1/type 1 like (Ty1; a 52 bp deletion; p.L367fs*46) and type 2/type 2 like (Ty2; a 5 bp TTGTC insertion; p.K385fs*47) CALR mut. Tefferi et al (Blood 2014;124:2465) reported that only Ty1 was prognostically favorable, while either the opposite or no effect was found in other studies (Leukemia 2015; 29:249). AIMS and METHODS. To evaluate the prognostic relevance of different types of CALR mut in a series of 396 PMF pts (2008 WHO criteria) seen in our center. CALR mut were analyzed by high-resolution capillary electrophoresis and confirmed by Sanger sequencing. Deep sequencing based on Ion Torrent PGM platform was used to analyze mutations in 17 genes previously shown to recur at discrete frequencies in chronic phase and leukemic transformation (LT) of PMF. Comparisons of quantitative variables between groups were carried out by the nonparametric Wilcoxon rank-sum test. RESULTS. 251 pts (63.4%) harbored JAK2 V617F mut, 21 (5.3%) MPL W515 mut, 74 (18.7%) CALR mut, of which 53 (71.6%) Ty1 and 21 (28.4%) Ty2; 50 pts (12.6%) were TN. There was no statistically significant difference between Ty1 and Ty2 for common hematologic and clinical variables. Conversely, both Ty1 and Ty2 differed from JAK2 V617F mut counterpart for younger age, lower leukocyte and higher platelet counts; males were more represented among Ty1 pts than other genotypes. Among CALR Ty1, more pts were in lower (low+Intermediate-1) IPSS risk categories (80%) compared to Ty2 (62%; P=0.04). With a median follow-up of 6.4 and 5.4 yrs for CALR mut Ty1 and Ty2 pts, respectively, there were less pts who died among Ty1 (17%) compared to Ty2 (47.6%) (P=0.007). Median survival of CALR Ty1 was 26.4 yr (range, 15.5-37.3) vs 7.4 yr (4.6-10.2) for CALR Ty2, 7.2 yr (5.7-8.6) for JAK2 V617F and 2.0 (1.6-2.4) for TN (P<.0001). The corresponding hazard ratio, taking CALR Ty1 pts as the reference, was 4.9 (95% CI, 1.8-12.9), 6.0 (95% CI, 2.7-13.4) and 20.6 (95% CI, 8.9-48.4) for CALR Ty2, JAK2 V617F and TN pts, respectively. In a multivariable Cox proportional hazard regression model, CALR Ty2 (HR 4.4, 95% CI 1.6-11.7), JAK2 V617F (HR 3.8, 95% CI 1.7-8.7) and TN (HR 9.2, 95%CI 3.8-22.2) all retained IPSS-independent prognostic impact on survival. Since some subclonal mutations are prognostically relevant in PMF (Leukemia 2013; 27:1861), we compared the mutational profile of 17 such genes, functionally clustered as epigenetic (DNMT3A, EZH2 TET2, IDH2, IDH1, ASXL1), of thespliceosome (SF3B1, SH2B3 U2AF1, SRSF2) orassociated with LT (cKIT, RUNX1, NRAS, KRAS, IKZF1, TP53 CBL) in 44 and 20 CALR mut Ty1 and Ty2 pts, respectively. There was a greater proportion of pts harboring at least one mutated gene among CALR Ty2 than Ty1 (70% vs 56.8%; P=0.02). Also, the proportion of pts with >2 mut was significantly greater in CALR mut Ty2 compared to Ty1 (40% vs 18%; P=0.01); in particular, 15% of CALR Ty2 mut pts had >3 mut compared to 2.2% in Ty1 (P=0.04). Frequency of individual mutations in Ty2 vs Ty1 was: epigenetic, 50% vs 50%; spliceasome 30% vs 4.5% (P=0.009); LT 30% vs 13.6% (P=0.06). Of note, SF3B1 mutations were 10-fold more represented in CALR mut Ty2 (25%) compared to Ty1 (2.2%; P<0.01). TP53 mutations were found in 10% of Ty2 and 0 in Ty 1. CONCLUSIONS. These findings support previous report that the prognostic advantage of CALR mutation in PMF regards only pts harboring type 1/type 1-like abnormalities. A greater mutational complexity involving subclonal mutations, particularly in genes of the spliceosome with an unusually high incidence of SF3B1 mutations, as well as increased number of mutated genes, may contribute to the dismal outcome of CALR mut Ty2 pts. Disclosures Vannucchi: Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Shire: Speakers Bureau.

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

Blood ◽  
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.

Integration of Mutations and Karyotype Towards a Genetics-Based Prognostic Scoring System (GPSS) for Primary Myelofibrosis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 406-406 ◽  
Author(s):  
Ayalew Tefferi ◽  
Paola Guglielmelli ◽  
Christy Finke ◽  
Terra L Lasho ◽  
Naseema Gangat ◽  
...  
Keyword(s):  
High Risk ◽  
Scoring System ◽  
Multivariable Analysis ◽  
Primary Myelofibrosis ◽  
World Health ◽  
Blast Transformation ◽  
Very High ◽  
Risk Categories

Abstract Background : Current prognostication in primary myelofibrosis (PMF) utilizes international prognostic scoring systems that rely on clinical parameters that are sensitive to day-to-day variations and subjective interpretation. Recent studies in PMF have disclosed important prognostic information attached to additional cytogenetic details (Blood. 2011;118:4595) and somatic mutations, including CALR and ASXL1 (NEJM. 2013;369:2379; Leukemia. 2013;27:1861). Methods : PMF diagnosis and definition of blast transformation (BT) were according to World Health Organization criteria (Blood. 2009;114:937). Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature (Cytogenetic and genome research. 2013. Prepublished on 2013/07/03 as DOI 10.1159/000353118). Previously published methods were used for analyses of CALR, JAK2, MPL and other prognostically-relevant mutations, including ASXL1, SRSF2, EZH2 and IDH(Leukemia. 2014;28:1472). Results : The training set included 964 Mayo Clinic patients (median age 65 years; 62% males) in whom informative karyotype or mutation information was available; cytogenetic information was available in 903 (94%) cases, JAK2/CALR/MPL mutational status in 532 (55%), ASXL1 in 425 (44%), SRSF2 in 434 (45%), IDH1/2 in 376 (39%) and EZH2 in 268 (28%). DIPSS-plus (JCO. 2011;29:392) risk distribution was high in 37% of patients, intermediate-2 in 37%, intermediate-1 in 15% and low in 11%. We used a revised risk stratification for cytogenetics (see accompanying ASH 2014 abstract) to distinguish four distinct cytogenetic risk categories: very high (monosomal karyotype, inv(3), i(17q), -7/7q-, 11q or 12p abnormalities; n=67), high (complex non-monosomal, two abnormalities not included in very high risk category, 5q-, +8, other autosomal trisomies except +9, and other sole abnormalities not included in other risk categories; n=164), intermediate (sole abnormalities of 20q-, 1q+ or any other sole translocation, and -Y or other sex chromosome abnormality; n=133) and low (normal or sole abnormalities of 13q- or +9; n=539). Mutational frequencies were 58% for JAK2, 25% CALR, 7% MPL, 36% ASXL1, 11% SRSF2, 5% IDH1/2 and 6% EZH2. The 131 cases with CALR mutations were further subclassified into two prognostically different groups: type 1/type 1-like (n=110) and type 2/type 2-like (n=21) (see accompanying ASH 2014 abstract). At a median follow-up time of 4.2 years for patients who are alive, 664 (69%) deaths and 70 BT (7%) were recorded. Age-adjusted multivariable analysis that included cytogenetic and mutational risk groups disclosed the following as independent predictors of shortened survival: very high risk karyotype (HR 4.2; 3 points), high risk karyotype (HR 1.9; 1 point), triple-negative (HR 2.8; 2 points), JAK2 (HR 3.1; 2 points), MPL (HR 3.1; 2 points), type 2/type 2-like CALR (HR 3.6; 2 points), ASXL1 (HR 1.9; 1 points) and SRSF2 (HR 1.9; 1 point); EZH2 (p=0.24) and IDH1/2 (p=0.68) and intermediate risk karyotype (p=0.87) were not significant. The above-mentioned significant variables and age demarcated at 60 years (2 points), were subsequently used to develop an HR-derived, genetics-based prognostic scoring system (GPSS) for 369 patients who were fully informative for both karyotype and all significant mutations: low risk (0 points; n=31), intermediate-1 (1 or 2 points; n=90), intermediate-2 (3 or 4 points; n=133) and high (5 or more points; n=115); the corresponding median survivals were >17, 9 (HR 4.7, 95% CI 1.7-13.0), 5 (HR 10.7, 95% CI 3.9-29.3) and 2.2 (HR 29.2, 95% CI 10.6-80.0) years (Figure 1). High risk GPSS was also associated with higher BT rate (HR 7.4, 95% CI 2.1-26.3). The prognostic distinction between high/intermediate-2 and low/intermediate-1 risk GPSS, in terms of both overall (median 5 vs 26.4 years; HR 7.1, 95% CI 3.3-14.9) and leukemia-free survival (median 11.6 years vs not reached; HR 9.4, 95% CI 2.2-41.0) was validated in an independent cohort of 183 patients from the University of Florence (Figure 2). Conclusions : The current study demonstrates the feasibility of genetics-based prognostic models in PMF that rely on objective parameters that are amenable to further refinement as new genetic information becomes available. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals A 27-Gene NGS Panel in Primary Myelofibrosis Identifies ASXL1, CBL, RUNX1 and SRSF2 Mutations As Being Unfavorable and Absence of Any Non-Driver Mutation As Being Favorable to Survival

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 350-350
Author(s):  
Ayalew Tefferi ◽  
Terra L. Lasho ◽  
Christy Finke ◽  
Yoseph Elala ◽  
Daniela Barraco ◽  
...  
Keyword(s):  
Triple Negative ◽  
Sequence Data ◽  
Hematologic Malignancy ◽  
Univariate Analysis ◽  
Multivariable Analysis ◽  
Primary Myelofibrosis ◽  
Driver Mutation ◽  
Driver Mutations ◽  
Prognostic Relevance ◽  
Mutational Status

Abstract Background : In primary myelofibrosis (PMF), ̴ 88% of patients harbor one of three "driver" mutations, with mutational frequencies of approximately 60%, 22% and 6% for JAK2, CALR and MPL, respectively. Other "non-driver" mutations have also been described in PMF and some of them and their number have been associated with inferior survival (Leukemia. 2014;28:1804). We applied next generation sequencing (NGS) with a broader panel of MPN-relevant genes, in order to identify additional mutations of prognostic relevance as well as obtain additional information regarding the prognostic value of 'number of mutations'. Methods: Targeted capture assays were carried out on bone marrow or whole blood DNA specimens obtained at time of referral for the following genes: TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, and SETBP1. Paired-end indexed libraries were prepared from individual patient DNA using the NEB Next Ultra Library prep protocol on the Agilent Bravo liquid handler (NEB, Ipswich, MA/Agilent Technologies Inc, Santa Clara, CA). Capture libraries were assembled according to Nimblegen standard library protocol (Roche Nimblegen, Inc, Basel, Switzerland). Base-calling was performed using Illumina's RTA version 1.17.21.3. Genesifter® software was utilized (PerkinElmer, Danvers, Massachusetts) to analyze targeted sequence data. Nucleotide variants were called using the Genome Analysis Toolkit (GATK-Broad Institute, Cambridge, MA). Specific variants were deemed as mutations if they are associated with a hematologic malignancy (as identified by COSMIC database), or if they have not been associated with a dbSNP. Results: 180 PMF patients were evaluated (median age 63 years; 65% males). DIPSS-plus risk distribution was 32% high, 38% intermediate-2, 17% intermediate-1 and 13% low. Driver mutation distribution was 62% JAK2, 22% CALR, 9% triple-negative and 7% MPL. Karyotype was abnormal in 41% of patients and unfavorable in 12%. Mutations other than JAK2, CALR or MPL (i.e. "non-driver" mutations) were seen in 150 (83%) patients including 88% of "triple-negative" cases. 62 (34%) patients harbored one, 55 (31%) two, 16 (9%) three and 17 (10%) four or more. Mutational frequencies were: ASXL1 36%, TET2 18%, SRSF2 17%, U2AF1 17%, ZRSR2 11%, SF3B1 10%, DNMT3A 9%, CEBPA (9%), Tp53 7%, SETBP1 6%, CBL 5%, IDH1/2 5%, SH2B3 4%, CSF3R 4%, NRAS 4%, RUNX1 3% and ≤2% for SUZ12, KIT, PTPN11, NPM1 and EZH2. DIPSS-plus high/intermediate-2 risk patients displayed higher number of mutations (p=0.0004) and higher mutational frequencies for ASXL1 (p=0.02), SRSF2 (p=0.004) and CBL (p=0.02). Associations noted included JAK2 with U2AF1 (p=0.03), unfavorable karyotype with CBL (p=0.01) and normal karyotype with ZRSR2 mutations (p=0.04). At a median follow-up of 4 years, 111 (62%) deaths were documented. For examination of impact on survival, we considered 'number of mutations' and specific mutations with >2% frequency. Accordingly, in univariate analysis, survival was adversely affected by 'number of mutations' (Figure 1) and presence of ASXL1, SRSF2, IDH1/2, U2AF1, RUNX1 and CBL mutations. For multivariable analysis, we considered three categories (zero, 1-3 and ≥4) for number of mutations based on the results from univariate analysis (Figure 1); the results showed ≥4 mutations, 1-3 mutations, RUNX1, CBL, ASXL1 and SRSF2 mutations were independently associated with shortened survival; the respective HR (95% CI) were 4 (1.4-11.1), 3 (1.3-6.8), 2.9 (1.1-8.1), 2.8 (1.3-6.3), 1.8 (1.2-2.7) AND 1.7 (1.03-2.7). When the multivariable analysis was repeated including only the 150 patients with at least one non-driver mutation, the 'number of mutations' was no longer significant (p=0.35) but ASXL1, CBL, RUNX1 and SRSF2 mutations retained their significance. The prognostic relevance of ASXL1 and CBL continued to be apparent even after the addition of DIPSS-plus and driver mutation profile to the multivariable model. Conclusions: Mutations other than JAK2, CALR or MPL occur in more than 80% of patients with PMF, including those with "triple-negative" driver mutational status. The absence of such mutations is independently favorable for survival while the prognostic effect of their presence is influenced by ASXL1, CBL, RUNX1 and SRSF2 mutations. Figure 1. Figure 1. Disclosures Pardanani: Stemline: Research Funding.

scholarly journals Update from the latest WHO classification of MPNs: a user’s manual

Hematology ◽  
2016 ◽  
Vol 2016 (1) ◽  
pp. 534-542 ◽  
Author(s):  
Francesco Passamonti ◽  
Margherita Maffioli
Keyword(s):  
Diagnostic Criteria ◽  
Who Classification ◽  
Triple Negative ◽  
Myeloproliferative Neoplasms ◽  
Primary Myelofibrosis ◽  
World Health ◽  
Driver Mutations ◽  
Health Organization

Abstract The 2016 multiparameter World Health Organization (WHO) classification for Philadelphia-negative myeloproliferative neoplasms (MPNs) integrates clinical features, morphology, and genetic data to diagnose polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The main novelties are: (1) the reduction of the hemoglobin (Hb) level threshold to diagnose PV, now established at 16.5 g/dL for men and 16 g/dL for women (based on the identification of MPN patients with PV-consistent bone marrow [BM] features and a Hb level lower than that established in the 2008 WHO classification for PV); (2) the recognition of prefibrotic/early PMF, distinguishable from ET on the basis of BM morphology, an entity having a higher tendency to develop overt myelofibrosis or acute leukemia, and characterized by inferior survival; (3) the central role of BM morphology in the diagnosis of ET, prefibrotic/early PMF, PMF, and PV with borderline Hb values; megakaryocyte number and morphology (typical in ET, atypical in both PMF forms) accompanied by a new distinction of reticulin fibrosis grade in PMF (grade 1 in prefibrotic/early PMF and grade 2-3 in PMF) constitute diagnostic criteria; and (4) the inclusion of all mutually exclusive MPN driver mutations (JAK2, CALR, and MPL) as major diagnostic criteria in ET and PMF; 10% to 15% of these patients are triple negative, and in these cases the search for an additional clonal marker (eg, mutations in ASXL1, EZH2, TET2, IDH1/IDH2, SRSF2, and SF3B1) is warranted.

Evaluation of Emergency Medical Team Coordination Following the 2015 Nepal Earthquake

2020 ◽  
pp. 1-8
Author(s):  
Yosuke Takada ◽  
Yasuhiro Otomo ◽  
Khem Bahadur Karki
Keyword(s):  
Online Survey ◽  
World Health ◽  
Medical Team ◽  
Team Coordination ◽  
Emergency Medical Team ◽  
Nepal Earthquake ◽  
Emergency Medical ◽  
Health Organization

ABSTRACT Objectives: After the Nepal earthquake in 2015, for the first time, the Emergency Medical Team Coordination Cell (EMTCC) was activated. This study aims to evaluate the emergency medical team (EMT) coordination in the aftermath of the Nepal earthquake in 2015. Methods: This is a retrospective study that (a) describes the coordination process in Nepal, and (b) reviews and analyzes the EMT database in Nepal to classify the EMTs based on the World Health Organization (WHO) EMT classification, an online survey for EMT coordination, and the Geographic Information System-analyzed EMT distribution. Results: We recorded 150 EMTs, which included 29 Type 1-Mobile, 71 Type 1-Fixed, 22 Type 2, 1 Type 3, and 27 specialist cell recorded EMTs including the military team. The EMTs were allocated based on the number of casualties in that area. The Type 1 EMTs were deployed around Type 2 EMTs. Conclusions: The EMT Classification is useful for the effective posting of EMTs. However, the method of onsite multi registration has room for improvement. The WHO should provide an opportunity for EMTCC training for better coordination of disasters.

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