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Loss-of-function TET2 mutations (TET2 MT ) are frequent early clonal events in myeloid neoplasms and are thought to confer a fitness advantage to hematopoietic precursors. This large, multi-institutional study (n = 1084), investigated the TET2 mutational landscape and prognostic implications of the number, type, and location of TET2 MT and the epistatic relationship with other somatic events in chronic myelomonocytic leukemia (CMML). Nine hundred and forty-two TET2 MT were identified in 604 (56%) patients, of which 710 (75%) were predicted to be truncating (involving the catalytic domain). Three hundred and sixteen (29%) patients had ≥1 TET2 MT , with 28%, 1%, and 0.2% harboring 2, 3, and 5 mutations, respectively. In comparison to TET2 WT , TET2 MT patients were older in age, more likely to have dysplastic CMML, a higher number of co-occurring mutations, and lower-risk stratification. Importantly, TET2 MT were associated with a survival advantage (49 vs. 30 months, p < 0.0001), especially in the context of multiple TET2 MT (≥2; 57 months, p < 0.001), and truncating TET2 MT (51 months, p < 0.001). In addition, the adverse prognostic impact of ASXL1 MT was partially mitigated by concurrent TET2 MT , with the ASXL1 WT /TET2 MT genotype having better outcomes and resulting in further risk stratification of ASXL1 inclusive CMML prognostic models, in comparison to ASXL1 MT alone.

International collaborations over the years have produced a series of prognostic models for primary myelofibrosis (PMF), including the recently unveiled mutation-enhanced international prognostic scoring systems for transplant-age patients (MIPSS70 and MIPSS70-plus). In the current study, we considered the feasibility of a genetically inspired prognostic scoring system (GIPSS) that is exclusively based on genetic markers. Among 641 cytogenetically annotated patients with PMF and informative for previously recognized adverse mutations, multivariable analysis identified "VHR" karyotype, "unfavorable" karyotype, absence of type 1/like CALR mutation and presence of ASXL1, SRSF2, or U2AF1Q157 mutation, as inter-independent predictors of inferior survival; the respective HRs (95% CI) were 3.1 (2.1-4.3), 2.1 (1.6-2.7), 2.1 (1.6-2.9), 1.8 (1.5-2.3), 2.4 (1.9-3.2), and 2.4 (1.7-3.3). Based on HR-weighted risk points, a four-tiered GIPSS model was devised: low (zero points; n = 58), intermediate-1 (1 point; n = 260), intermediate-2 (2 points; n = 192), and high (≥3 points; n = 131); the respective median (5-year) survivals were 26.4 (94%), 8.0 (73%), 4.2 (40%), and 2 (14%) years; the model was internally validated by bootstrapping and its predictive accuracy was shown to be comparable to that of MIPSS70-plus. GIPPS offers a low-complexity prognostic tool for PMF that is solely dependent on genetic risk factors and, thus, forward-looking in its essence.

In a cohort of 466 patients, we sought to clarify the prognostic relevance of ASXL1 and SETBP1 mutations, among others, in World Health Organization-defined chronic myelomonocytic leukemia (CMML) and its added value to the Mayo prognostic model. In univariate analysis, survival was adversely affected by ASXL1 (nonsense and frameshift) but not SETBP1 mutations. In multivariable analysis, ASXL1 mutations, absolute monocyte count >10 × 10(9)/l, hemoglobin <10 g/dl, platelets <100 × 10(9)/l and circulating immature myeloid cells were independently predictive of shortened survival: hazard ratio (95% confidence interval (CI)) values were 1.5 (1.1-2.0), 2.2 (1.6-3.1), 2.0 (1.6-2.6), 1.5 (1.2-1.9) and 2.0 (1.4-2.7), respectively. A regression coefficient-based prognostic model based on these five risk factors delineated high (≥3 risk factors; HR 6.2, 95% CI 3.7-10.4) intermediate-2 (2 risk factors; HR 3.4, 95% CI 2.0-5.6) intermediate-1 (one risk factor; HR 1.9, 95% CI 1.1-3.3) and low (no risk factors) risk categories with median survivals of 16, 31, 59 and 97 months, respectively. Neither ASXL1 nor SETBP1 mutations predicted leukemic transformation. The current study confirms the independent prognostic value of nonsense/frameshift ASXL1 mutations in CMML and signifies its added value to the Mayo prognostic model, as had been shown previously in the French consortium model.

Calreticulin (CALR) mutations were recently described in JAK2 and MPL unmutated primary myelofibrosis (PMF) and essential thrombocythemia. In the current study, we compared the clinical, cytogenetic and molecular features of patients with PMF with or without CALR, JAK2 or MPL mutations. Among 254 study patients, 147 (58%) harbored JAK2, 63 (25%) CALR and 21 (8.3%) MPL mutations; 22 (8.7%) patients were negative for all three mutations, whereas one patient expressed both JAK2 and CALR mutations. Study patients were also screened for ASXL1 (31%), EZH2 (6%), IDH (4%), SRSF2 (12%), SF3B1 (7%) and U2AF1 (16%) mutations. In univariate analysis, CALR mutations were associated with younger age (P<0.0001), higher platelet count (P<0.0001) and lower DIPSS-plus score (P=0.02). CALR-mutated patients were also less likely to be anemic, require transfusions or display leukocytosis. Spliceosome mutations were infrequent (P=0.0001) in CALR-mutated patients, but no other molecular or cytogenetic associations were evident. In multivariable analysis, CALR mutations had a favorable impact on survival that was independent of both DIPSS-plus risk and ASXL1 mutation status (P=0.001; HR 3.4 for triple-negative and 2.2 for JAK2-mutated). Triple-negative patients also displayed inferior LFS (P=0.003). The current study identifies 'CALR(-)ASXL1(+)' and 'triple-negative' as high-risk molecular signatures in PMF.

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F- or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with ruxolitinib in suppressing the growth of JAK2V617F-mutant SET2 cells. Importantly, MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.

We evaluated the prognostic relevance of several clinical and laboratory parameters in 226 Mayo Clinic patients with chronic myelomonocytic leukemia (CMML): 152 (67%) males and median age 71 years. At a median follow-up of 15 months, 166 (73%) deaths and 33 (14.5%) leukemic transformations were documented. In univariate analysis, significant risk factors for survival included anemia, thrombocytopenia, increased levels of white blood cells, absolute neutrophils, absolute monocyte count (AMC), absolute lymphocytes, peripheral blood and bone marrow blasts, and presence of circulating immature myeloid cells (IMCs). Spliceosome component (P=0.4) and ASXL1 mutations (P=0.37) had no impact survival. On multivariable analysis, increased AMC (>10 × 10(9)/l, relative risk (RR) 2.5, 95% confidence interval (CI) 1.7-3.8), presence of circulating IMC (RR 2.0, 95% CI 1.4-2.7), decreased hemoglobin (<10 g/dl, RR 1.6, 99% CI 1.2-2.2) and decreased platelet count (<100 × 10(9)/l, RR 1.4, 99% CI 1.0-1.9) remained significant. Using these four risk factors, a new prognostic model for overall (high risk, RR 4.4, 95% CI 2.9-6.7; intermediate risk, RR 2.0, 95% CI 1.4-2.9) and leukemia-free survival (high risk, RR 4.9, 95% CI 1.9-12.8; intermediate risk, RR 2.6, 95% CI 1.1-5.9) performed better than other conventional risk models and was validated in an independent cohort of 268 CMML patients.

Isocitrate dehydrogenase (IDH) mutations are frequent in blast-phase myeloproliferative neoplasms and might therefore contribute to leukemic transformation. We examined this possibility in 301 consecutive patients with chronic-phase primary myelofibrosis (PMF). The mutant IDH was detected in 12 patients (4%): 7 IDH2 (5 R140Q, 1 R140W and 1 R172G) and 5 IDH1 (3 R132S and 2 R132C). In all, 6 (50%) of the 12 IDH-mutated patients also expressed JAK2V617F. Overall, 18 (6%) patients displayed only MPL and 164 (54.3%) only JAK2 mutations. Multivariable analysis that accounted for conventional risk factors disclosed inferior overall survival (OS; P=0.03) and leukemia-free survival (LFS; P=0.003) in IDH-mutated patients: OS hazard ratio (HR) was 0.39 (95% confidence interval (95% CI) 0.2-0.75), 0.50 (95% CI 0.27-0.95) and 0.53 (95% CI 0.23-1.2) for patients with no, JAK2 or MPL mutations, respectively. Further analysis disclosed a more pronounced effect for the mutant IDH on OS and LFS in the presence (P=0.0002 and P<0.0001, respectively) as opposed to the absence (P=0.34 and P=0.64) of concomitant JAK2V617F. Analysis of paired samples obtained during chronic- and blast-phase disease revealed the presence of both IDH and JAK2 mutations at both time points. Our observations suggest that IDH mutations in PMF are independent predictors of leukemic transformation and raise the possibility of leukemogenic collaboration with JAK2V617F.

Unlike the case with acute myeloid leukemia, there is limited information on the prognostic impact of isocitrate dehydrogenase (IDH) mutations in myelodysplastic syndromes (MDS). In the current study of 277 patients with MDS, IDH mutations were detected in 34 (12%) cases: 26 IDH2 (all R140Q) and 8 IDH1 (6 R132S and 2 R132C). Mutational frequency was 4% (2 of 56) in refractory anemia with ring sideroblasts, 12% (16 of 130) in refractory cytopenia with multilineage dysplasia, 14% (2 of 14) in MDS-unclassifiable, 14% (6 of 42) in refractory anemia with excess blasts (RAEB)-1 and 23% (8 of 35) in RAEB-2. Normal karyotype was noted in all but one IDH1-mutated cases and 13 IDH2-mutated cases. Multivariable analysis identified presence of mutant IDH1 (P=0.0004; hazard ration 4.0, 95% confidence interval 1.9-8.8), revised International Prognostic Scoring System risk category (P<0.0001), and red cell transfusion need (P=0.002) as independent predictors of inferior survival. In a similar multivariable analysis, mutant IDH1 was the only variable associated with shortened leukemia-free survival (P=0.001; hazard ration 7.0, 95% confidence interval 2.3-20.8). The presence of IDH2R140Q did not affect the overall (P=0.54) or leukemia-free (P=0.81) survival. The current study suggests a powerful adverse prognostic effect for mutant IDH1 in MDS.

The 2008 World Health Organization (WHO) criteria were used to identify 88 consecutive Mayo Clinic patients with 'myelodysplastic syndrome with isolated del(5q)' (median age 74 years; 60 females). In all, 60 (68%) patients were followed up to the time of their death. Overall median survival was 66 months; leukemic transformation was documented in five (5.7%) cases. Multivariable analysis identified age >or=70 years (P=0.01), transfusion need at diagnosis (P=0.04) and dysgranulopoiesis (P=0.02) as independent predictors of shortened survival; the presence of zero (low risk), one (intermediate risk) or >or=2 (high risk) risk factors corresponded to median survivals of 102, 52 and 27 months, respectively. Janus kinase 2 (JAK2), thrombopoietin receptor (MPL), isocitrate dehydrogenase 1 (IDH1) and IDH2 mutational analysis was performed on archived bone marrows in 78 patients; JAK2V617F and MPLW515L mutations were shown in five (6.4%) and three (3.8%) patients, respectively, and did not seem to affect phenotype or prognosis. IDH mutations were not detected. Survival was not affected by serum ferritin and there were no instances of death directly related to iron overload. The current study is unique in its strict adherence to WHO criteria for selecting study patients and providing information on long-term survival, practical prognostic factors, baseline risk of leukemic transformation and the prevalence of JAK2, MPL and IDH mutations.

In a multi-institutional collaborative project, 1473 patients with myeloproliferative neoplasms (MPN) were screened for isocitrate dehydrogenase 1 (IDH1)/IDH2 mutations: 594 essential thrombocythemia (ET), 421 polycythemia vera (PV), 312 primary myelofibrosis (PMF), 95 post-PV/ET MF and 51 blast-phase MPN. A total of 38 IDH mutations (18 IDH1-R132, 19 IDH2-R140 and 1 IDH2-R172) were detected: 5 (0.8%) ET, 8 (1.9%) PV, 13 (4.2%) PMF, 1 (1%) post-PV/ET MF and 11 (21.6%) blast-phase MPN (P<0.01). Mutant IDH was documented in the presence or absence of JAK2, MPL and TET2 mutations, with similar mutational frequencies. However, IDH-mutated patients were more likely to be nullizygous for JAK2 46/1 haplotype, especially in PMF (P=0.04), and less likely to display complex karyotype, in blast-phase disease (P<0.01). In chronic-phase PMF, JAK2 46/1 haplotype nullizygosity (P<0.01; hazard ratio (HR) 2.9, 95% confidence interval (CI) 1.7-5.2), but not IDH mutational status (P=0.55; HR 1.3, 95% CI 0.5-3.4), had an adverse effect on survival. This was confirmed by multivariable analysis. In contrast, in both blast-phase PMF (P=0.04) and blast-phase MPN (P=0.01), the presence of an IDH mutation predicted worse survival. The current study clarifies disease- and stage-specific IDH mutation incidence and prognostic relevance in MPN and provides additional evidence for the biological effect of distinct JAK2 haplotypes.

Bone marrow DNA was screened for isocitrate dehydrogenase (IDH) mutations in 200 patients with chronic (n=166) or blast (n=34) phase myeloproliferative neoplasms (MPN). Included among the former were 77 patients with primary myelofibrosis (PMF), 47 essential thrombocythemia and 38 polycythemia vera (PV). Nine IDH mutations (5 IDH1 and 4 IDH2) were detected; mutational frequencies were approximately 21% (7 of 34) for blast-phase MPN and approximately 4% (3 of 77) for PMF. IDH mutations were seen in only 1 of 12 paired chronic-blast-phase samples and in none of 27 concurrently studied acute myeloid leukemia (AML) patients without antecedent MPN. IDH1 mutations included R132C (n=4; two post-PMF AML, one post-PV AML and one PMF) and R132S (n=1; post-PMF AML). IDH2 mutations included R140Q (n=3; one post-PMF AML, one post-PV AML and one PMF) and a novel R140W (n=1; mutation found in both chronic- and blast-phase samples). The entire study cohort was also screened for JAK2 and MPL mutations and JAK2V617F was found in three IDH-mutated cases (two PMF and one PV). This study shows a relatively high incidence of IDH mutations in blast-phase MPN, regardless of JAK2 mutational status, and the occurrence of similar mutations in chronic-phase PMF.

The germline JAK2 haplotype 46/1, tagged by the 'C' allele of single-nucleotide polymorphism (SNP) rs12343867 (C/T), has been associated with JAK2V617F (VF)-positive myeloproliferative neoplasms. SNP rs12343867 was genotyped using bone marrow DNA in 226 consecutive patients with essential thrombocythemia (ET) with concomitant analysis of VF allele burden. The incidence of the 46/1-linked C allele was significantly higher in ET (genotype: CC 15%, CT 52%, TT 33%; C-allele frequency: 41%) than in population controls (P<0.01). Genotype distributions were similar among VF-positive/VF-negative patients (genotype: CC 18/11%, CT 52/53%, TT 30/36%; C-allele: 44/38%; P=0.29). Haplotype 46/1 frequency was remarkably similar when comparing VF-negative patients to those with <10% VF allele burden, but significantly higher in the presence of >10% VF allele burden (genotype: CC 11/13/38%, CT 53/56/38%, TT 36/31/24%; C-allele frequency: 38/41/57%; P<0.01). The clinical features of 46/1-positive and -negative ET were indistinguishable, including blood counts, rate of thrombosis/disease transformation and survival. We conclude that JAK2 haplotype 46/1 confers susceptibility to developing ET independent of VF mutational status and does not seem to further affect the clinical phenotype or prognosis.

A common JAK2 germline haplotype (46/1) has been associated with JAK2V617F (VF)-positive myeloproliferative neoplasms. The rs12343867 SNP (C/T) tags this haplotype. A total of 130 patients (77 VF-positive) with primary myelofibrosis (PMF) were analyzed for this informative SNP, using bone marrow-derived DNA. The observed 46/1 C allele frequencies in VF-positive (50%) and VF-negative (36%) patients were both significantly higher than expected in population controls (P<0.01). Genotype distributions in VF-positive/VF-negative patients were CC 31%/9%, CT 38%/53% and TT 31%/38% (P=0.01). CC genotype/C-allele frequencies in patients with <20% VF mutation burden (12%/37%) were similar (P=0.95) to those seen in VF-negative patients (9%/36%), but were significantly lower (P<0.01) than those seen in the presence of >50% mutation burden ( approximately 67%/71%). The rs12343867 genotype did not correlate with the International Prognostic Scoring System (IPSS) score or karyotype. Unexpectedly, the TT genotype was associated with shortened survival (P<0.01), which was not accounted for by IPSS score or VF allele burden. We conclude that JAK2 germline genetic variation affects disease susceptibility, and possibly survival, in PMF, regardless of VF mutational status. Allelic distortion from acquired uniparental disomy contributes to the appearance of a more pronounced effect on disease susceptibility in VF-positive patients, when studying clonally affected tissue.

TET2 (TET oncogene family member 2) is a candidate tumor suppressor gene located at chromosome 4q24, and was recently reported to be mutated in approximately 14% of patients with JAK2V617F-positive myeloproliferative neoplasms. We used high-throughput DNA sequence analysis to screen for TET2 mutations in bone marrow-derived DNA from 48 patients with systemic mastocytosis (SM), including 42 who met the 2008 WHO (World Health Organization) diagnostic criteria for SM and 6 with FIP1L1-PDGFRA. Twelve (29%) SM, but no FIP1L1-PDGFRA patients, had TET2 mutations. A total of 17 mutations (13 frameshift, 2 nonsense and 2 missense) were documented in 2 (15%) of 13 indolent SM patients, 2 (40%) of 5 aggressive SM, and 8 (35%) of 23 SM associated with a clonal non-mast cell-lineage hematopoietic disease (P=0.52). KITD816V was detected by PCR sequencing in 50 or 20% of patients with or without TET2 mutation (P=0.05), respectively. Multivariable analysis showed a significant association between the presence of TET2 mutation and monocytosis (P=0.0003) or female sex (P=0.05). The association with monocytosis was also observed in non-indolent SM (n=29), in which the presence of mutant TET2 did not affect survival (P=0.98). We conclude that TET2 mutations are frequent in SM, segregate with KITD816V and influence phenotype without necessarily altering prognosis.

High-throughput DNA sequence analysis was used to screen for TET2 mutations in bone marrow-derived DNA from 239 patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs). Thirty-two mutations (19 frameshift, 10 nonsense, 3 missense; mostly involving exons 4 and 12) were identified for an overall mutational frequency of approximately 13%. Specific diagnoses included polycythemia vera (PV; n=89), essential thrombocythemia (ET; n=57), primary myelofibrosis (PMF; n=60), post-PV MF (n=14), post-ET MF (n=7) and blast phase PV/ET/MF (n=12); the corresponding mutational frequencies were approximately 16, 5, 17, 14, 14 and 17% (P=0.50). Mutant TET2 was detected in approximately 17 and approximately 7% of JAK2V617F-positive and -negative cases, respectively (P=0.04). However, this apparent clustering of the two mutations was accounted for by an independent association between mutant TET2 and advanced age; mutational frequency was approximately 23% in patients > or =60 years old versus approximately 4% in younger patients (P<0.0001). The presence of mutant TET2 did not affect survival, leukemic transformation or thrombosis in either PV or PMF; a correlation with hemoglobin <10 g per 100 ml in PMF was noted (P=0.05). We conclude that TET2 mutations occur in both JAK2V617F-positive and -negative MPN, are more prevalent in older patients, display similar frequencies across MPN subcategories and disease stages, and hold limited prognostic relevance.