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JAK2 mutations in myeloproliferative neoplasms (MPNs) are associated with the germline GGCC (46/1) haplotype. In 2010, we reported an association between shortened survival in primary myelofibrosis (PMF) and nullizygosity for the JAK2 46/1 haplotype. In the current study, we have increased the number of informative cases from 130 to 414 (median age 63 years; 63% males), in order to revisit with the phenotypic and prognostic relevance of the JAK2 46/1 haplotype in PMF. 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 = .02). 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), most pronounced in JAK2 (P <.001), as opposed to CALR/MPL mutated (P = .48) or triple-negative cases (P = .27). Multivariable analysis that included karyotype, driver mutational status and high-molecular risk mutations confirmed the independent prognostic contribution of nullizygosity for the 46/1 haplotype (P = .02; HR 1.4, 95% CI 1.1-1.8). Nullizygosity for 46/1 also remained significant in the context of the genetically-inspired GIPSS risk model (P = .04), but not in the context of the integrated genetics-clinical MIPSS70+ version 2.0 model (P = .4). Leukemia-free survival was not affected by the 46/1 haplotype (P = .6). The current study confirms the association of nullizygosity for the JAK2 GGCC (46/1) haplotype with inferior survival in JAK2-mutated PMF., (© 2018 Wiley Periodicals, Inc.)

In the last decade, several prognostic models for primary myelofibrosis (PMF) have been introduced and shown to be effective in predicting overall survival. The main objective for this study was to identify clinical and genetic markers of very long (20+ years) survival in PMF. A total of 1282 patients with PMF were considered (median age 65 years, range 19-92; 63% males); 26 (2%) patients (median age 51 years, range 28-71; 38% males) survived their disease for at least 20 years (long-lived patients) and 626 (49%) patients (median age 68 years, range 27-92; 66% males) died within 5 years of their diagnosis (short-lived patients). Multivariable logistic regression analysis identified 7 variables that were associated with survival beyond 20 years: age ≤ 70 years (P = .002); female sex (P = .03); hemoglobin level ≥ 10 g/dL for women and ≥ 11 g/dL for men (P = .03), leukocyte count ≤25 × 10 9 /L (P = .009), platelet count ≥100 × 10 9 /L (P = .002), circulating blasts <2% (P = .03) and absence of constitutional symptoms (P = .04). Five-year mortality was independently predicted by high-molecular risk mutations (P < .001); unfavorable or very high risk karyotype (P < .001); absence of type 1/like CALR mutation (P < .001); age > 70 years (P < .001); constitutional symptoms (P < .001); hemoglobin level < 10 g/dL for women and < 11 g/dL for men (P < .001); leukocyte count >25 × 10 9 /L (P = .004); and circulating blasts ≥2% (P = .001). This study suggests that genetic risk factors in PMF are associated with early mortality while survival beyond 20 years could be predicted by easily accessible clinical variables, including age, sex, blood counts, and symptoms., (© 2018 Wiley Periodicals, Inc.)

Among 1306 patients with primary myelofibrosis (PMF), we sought to identify risk factors that predicted leukemic transformation (LT) in the first 5 years of disease and also over the course of the disease. 149 (11%) LT were documented; patients who subsequently developed LT (n = 149), compared to those who remained in chronic phase disease (n = 1,157), were more likely to be males (p = 0.02) and display higher circulating blasts (p = 0.03), ASXL1 (p = 0.01), SRSF2 (p = 0.001) and IDH1 (p = 0.02) mutations. Logistic regression analysis identified IDH1, ASXL1 and SRSF2 mutations, very high-risk karyotype, age > 70 years, male sex, circulating blasts ≥ 3%, presence of moderate or severe anemia and constitutional symptoms, as predictors of LT in the first 5 years of diagnosis. Time-to-event Cox analysis confirmed LT prediction for IDH1 mutation (HR 4.3), circulating blasts ≥ 3% (HR 3.3), SRSF2 mutation (HR 3.0), age > 70 years (HR 2.1), ASXL1 mutation (HR 2.0) and presence of moderate or severe anemia (HR 1.9). HR-based risk point allocation resulted in a three-tiered LT risk model: high-risk (LT incidence 57%; HR 39.3, 95% CI 10.8-114), intermediate-risk (LT incidence 17%; HR 4.1, 95% CI 2.4-7.3) and low-risk (LT incidence 8%). The current study provides a highly discriminating LT predictive model for PMF.

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.

The 2013 discovery of calreticulin (CALR) mutations in myeloproliferative neoplasms was attended by their association with longer survival in primary myelofibrosis (PMF). Subsequent studies have suggested prognostic distinction between type 1/like and type 2/like CALR mutations and detrimental effect from triple-negative mutational status. Among 709 Mayo Clinic patients with PMF, 467 (66%) harbored JAK2, 112 (16%) CALR type 1/like, 24 (3.4%) CALR type 2/like, 38 (5.4%) MPL mutations and 68 (10%) were triple-negative. Survival was longer with type 1/like CALR, compared to JAK2 (HR 2.6, 95% CI 1.9-3.5), type 2/like CALR (HR 2.5, 95% CI 1.4-4.5), MPL (HR 1.8, 95% CI 1.1-2.9) and triple-negative mutational status (HR 2.4, 95% CI 1.6-3.6), but otherwise similar between the non-type 1/like CALR mutational states (P = .41). In multivariable analysis, the absence of type 1/like CALR (P < .001; HR 2, 95% CI 1.4-2.7), presence of ASXL1/SRSF2 mutations (P < .001; HR 1.9, 95% CI 1.5-2.4) and DIPSS-plus (P < .001) were each predictive of inferior survival. Furthermore, among 210 patients with ASXL1/SRSF2 mutations, survival was significantly longer in the presence vs. absence of type 1/like CALR mutations (median 5.8 vs. 2.9 years; P < .001). Triple-negative status did not disclose additional prognostic information for overall or leukemia-free survival. The observations regarding the prognostic distinction between CALR mutation variants were validated in an external cohort of 386 patients from the University of Florence Careggi hospital. We conclude that type 1/like CALR mutations in PMF not only predict superior survival, but also partially amend the detrimental effect of high molecular risk mutations., (© 2017 Wiley Periodicals, Inc.)

Primary myelofibrosis (PMF)-associated pruritus is often severe and requires treatment. Fifty-one patients with bone marrow-proven PMF with associated pruritus were identified from a primary cohort of patients with PMF (n = 566) seen at our institution. We conducted a retrospective review of the clinical characteristics, severity of pruritus, type of treatment, and response of these patients. Thirty-two out of 51 patients (63 %) reported severe PMF-associated pruritus and required a total of 108 treatment episodes, with complete response (CR), partial response (PR) and no response (NR) observed in 22, 23, and 55 % of episodes, respectively. The most common treatment categories included JAK inhibitors (n = 19), anti-depressants (n = 18), and antihistamines (n = 17). Highest CR rates were observed in patients treated with a JAK inhibitor (53 %) and immunomodulatory drugs (IMiDS (50 %)). Emerging targeted therapies may result in better symptom control and higher response rates in patients suffering from severe PMF-associated pruritus.

Background: Current drugs for myeloproliferative neoplasm-associated myelofibrosis, including Janus kinase (JAK) inhibitors, do not induce complete or partial remissions. Imetelstat is a 13-mer lipid-conjugated oligonucleotide that targets the RNA template of human telomerase reverse transcriptase., Methods: We sought to obtain preliminary information on the therapeutic activity and safety of imetelstat in patients with high-risk or intermediate-2-risk myelofibrosis. Imetelstat was administered as a 2-hour intravenous infusion (starting dose, 9.4 mg per kilogram of body weight) every 1 to 3 weeks. The primary end point was the overall response rate, and the secondary end points were adverse events, spleen response, and independence from red-cell transfusions., Results: A total of 33 patients (median age, 67 years) met the eligibility criteria; 48% had received prior JAK inhibitor therapy. A complete or partial remission occurred in 7 patients (21%), with a median duration of response of 18 months (range, 13 to 20+) for complete responses and 10 months (range, 7 to 10+) for partial responses. Bone marrow fibrosis was reversed in all 4 patients who had a complete response, and a molecular response occurred in 3 of the 4 patients. Response rates were 27% among patients with a JAK2 mutation versus 0% among those without a JAK2 mutation (P=0.30) and 32% among patients without an ASXL1 mutation versus 0% among those with an ASXL1 mutation (P=0.07). The rate of complete response was 38% among patients with a mutation in SF3B1 or U2AF1 versus 4% among patients without a mutation in these genes (P=0.04). Responses did not correlate with baseline telomere length. Treatment-related adverse events included grade 4 thrombocytopenia (in 18% of patients), grade 4 neutropenia (in 12%), grade 3 anemia (in 30%), and grade 1 or 2 elevation in levels of total bilirubin (in 12%), alkaline phosphatase (in 21%), and aspartate aminotransferase (in 27%)., Conclusions: Imetelstat was found to be active in patients with myelofibrosis but also had the potential to cause clinically significant myelosuppression. (Funded by Geron; ClinicalTrials.gov number, NCT01731951.).

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.

Among spliceosome component mutations, those involving SF3B1 are most frequent in myelodysplastic syndromes with ring sideroblasts (MDS-RS; ∼ 75% incidence) and SRSF2 in chronic myelomonocytic leukemia (∼ 28% incidence). We recently reported on the lack of prognostic significance for SF3B1 mutations in both MDS-RS and primary myelofibrosis (PMF). In the current study, we examined the prevalence and prognostic relevance of SRSF2 mutations in PMF. Among 187 patients screened, 32 (17%) harbored SRSF2 monoallelic mutations affecting residue P95. Significant associations were demonstrated between SRSF2 mutations and advanced age (P < .01), IDH mutations (P < .01), and higher DIPSS-plus risk category (P = .03). SRSF2 mutations were associated with shortened overall (P < .01) and leukemia-free (P < .01) survival; the adverse effect on survival was independent of DIPSS-plus (P = .01; HR = 1.9; 95% CI, 1.1-3.0) and IDH mutations (P < .01; HR = 2.3; 95% CI, 1.4-3.8). In conclusion, SRSF2 mutations are relatively common in PMF, cluster with IDH mutations, and are independently predictive of poor outcome.

Plasma cytokine milieu is abnormal in primary myelofibrosis (PMF) and correlates with disease phenotype and prognosis. In this study, we show that several plasma cytokines are also abnormally expressed in polycythemia vera (PV; n = 65), compared to normal controls (n = 35), but with a significantly different pattern than that of PMF (n = 127). Direct phenotypic correlation in PV included levels of IL-12 with hematocrit; IL-1b, IL-2, IL-7, FGF-b, and HGF with leukocytosis; and IFN-α and IFN-γ with thrombocytosis. In univariate analysis, levels of 13 cytokines (out of 30 analyzed) correlated with survival but only MIP-1β remained significant on multivariable analysis that included the other cytokines as covariates. Increased level of MIP-1β (P < 0.01), older age (P < 0.01), and leukocytosis (P = 0.03) maintained their association with shortened survival, on multivariable analysis. This study provides preliminary observations that warrant a larger scale study and suggests the value of plasma cytokines as prognostic biomarkers in PV., (Copyright © 2012 Wiley Periodicals, Inc.)

Purpose: We hypothesized that surrogate markers of host immune response may predict survival in myeloid malignancies. Because of immediate practical applicability, we chose plasma immunoglobulin free light chain (FLC) concentration as the biomarker of interest., Patients and Methods: Two independent cohorts of patients with primary myelofibrosis (PMF) or myelodysplastic syndromes (MDS) were studied. Kappa (κ) and lambda (λ) FLCs were measured by a quantitative nephelometric assay. Patients with monoclonal FLC were excluded., Results: Values that were above the upper limit of normal for κ or λ FLC were documented in 33% of 240 patients with PMF and 46% of 74 patients with MDS. Increased FLC was significantly associated with increased creatinine, and advanced age in PMF (P < .001) and hemoglobin less than 10 g/dL in MDS (P = .005). In multivariable analysis, increased FLC predicted shortened survival in both PMF and MDS, independent of age, creatinine, and other conventional risk factors. Cutoff levels based on receiver operating characteristic analysis for κ plus λ total FLCs delineated risk groups with highly significant differences in overall survival; International Prognostic Scoring System-adjusted hazard ratio in PMF was 1.9 (95% CI, 1.3 to 2.7), and was 6.3 (95% CI, 2.7 to 16.6) in MDS. No correlations were seen with leukemia-free survival, karyotype, or JAK2, MPL, or IDH mutations. In patients with PMF who were studied by cytokine profiling, the prognostic value of an increased FLC level was independent of that from circulating interleukin-2 receptor (IL-2R) or IL-8 levels., Conclusion: Increased plasma FLC concentration predicts inferior survival in both PMF and MDS. Its lack of correlation with leukemia-free survival and tumor-specific genetic markers suggests a primarily host-driven biologic phenomenon that might be more broadly applicable.

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.

Recent clinical trials with JAK or mammalian target of rapamycin (mTOR) inhibitors in primary myelofibrosis (PMF) have identified pruritus as one of the most treatment-responsive disease traits. However, little is known about the prevalence of pruritus in PMF or its clinical and laboratory correlates. Among 566 consecutive patients with PMF seen at our institution, the presence or absence of pruritus was documented in 90 (16%) and 146 (26%) patients, respectively. Patients with pruritus were less likely to express MPLW515 (0% vs. 10%; P = 0.02) or leukopenia (8% vs. 24%; P = 0.002). The latter association was more pronounced in the absence of JAK2 or MPL mutations. Pruritus also clustered with marked leukocytosis (23% vs. 11%; P = 0.01) and JAK2V617F (71% vs. 59%; P = 0.08). Pruritus did not correlate with karyotype (P = 0.33), risk category per the Dynamic International Prognostic Scoring System (DIPSS)-plus (P = 0.37), DIPSS-plus-adjusted survival (P = 0.41), or leukemic transformation (P = 0.13). Plasma levels of 20 cytokines, which are known to be abnormally expressed in PMF, including IL-1b, IL-2R, IL-6, IL-8, and VEGF, were measured in 63 informative cases and showed no correlations with history of pruritus. We conclude that pruritus is relatively frequent in PMF and is prognostically irrelevant. The pathogenesis of PMF-associated pruritus is not necessarily linked to proinflammatory cytokines but may instead involve molecules that are either granulocyte-derived or influence granulopoiesis. The apparently differential effect of MPL vs. JAK2 mutations on pruritus requires further investigation., (Copyright © 2011 Wiley-Liss, Inc.)

Objective: To share our decades of experience with primary myelofibrosis and underscore the importance of outcomes research studies in designing clinical trials and interpreting their results., Patients and Methods: One thousand consecutive patients with primary myelofibrosis seen at Mayo Clinic between November 4, 1977, and September 1, 2011, were considered. The International Prognostic Scoring System (IPSS), dynamic IPSS (DIPSS), and DIPSS-plus were applied for risk stratification. Separate analyses were included for patients seen at time of referral (N=1000), at initial diagnosis (N=340), and within or after 1 year of diagnosis (N=660)., Results: To date, 592 deaths and 68 leukemic transformations have been documented. Parameters at initial diagnosis vs time of referral included median age (66 vs 65 years), male sex (61% vs 62%), red cell transfusion need (24% vs 38%), hemoglobin level less than 10 g/dL (38% vs 54%), platelet count less than 100 × 10(9)/L (18% vs 26%), leukocyte count more than 25 × 10(9)/L (13% vs 16%), marked splenomegaly (21% vs 31%), constitutional symptoms (29% vs 34%), and abnormal karyotype (31% vs 41%). Mutational frequencies were 61% for JAK2V617F, 8% for MPLW515, and 4% for IDH1/2. DIPSS-plus risk distributions at time of referral were 10% low, 15% intermediate-1, 37% intermediate-2, and 37% high. The corresponding median survivals were 17.5, 7.8, 3.6, and 1.8 years vs 20.0, 14.3, 5.3, and 1.7 years for patients younger than 60 years of age. Compared with both DIPSS and IPSS, DIPSS-plus showed better discrimination among risk groups. Five-year leukemic transformation rates were 6% and 21% in low- and high-risk patients, respectively., Conclusion: The current document should serve as a valuable resource for patients and physicians and provides context for the design and interpretation of clinical trials., (Copyright © 2012 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.)

MPL and JAK2V617F mutation analysis was performed in 603 patients with primary myelofibrosis (PMF) seen at the Mayo Clinic, USA (n=329) or University of Florence, Italy (n=274). Mutant MPL was detected in 49 (8.1%) patients and JAK2V617F in 350 (58%); 4 patients showed both mutations. MPLW515L/K was the commonest mutation; 2 patients showed novel mutations (L513ins and Q516-P518insAAAA). The US and Italy patient cohorts were separately analyzed for comparison of survival and clinical features between MPL-mutated, JAK2-mutated and JAK2/MPL-unmutated cases. JAK2/MPL-unmutated patients were significantly younger than their JAK2-mutated counterparts, in both patient cohorts (P<0.01). In the Florence only cohort, the presence of mutant MPL was associated with older age (P<0.01) and constitutional symptoms (P=0.04) and JAK2V617F with higher hemoglobin (P<0.01) and leukocyte (P=0.03) count; neither patient cohort showed significant associations with platelet count, hemoglobin <10 g/dl, abnormal/unfavorable karyotype, spleen size or prognostic score distribution. To date, 240 deaths and 79 leukemic transformations have been documented among all 603 study patients. Multivariable analysis disclosed no significant difference in overall or leukemia-free survival between the three molecular subgroups. We conclude that the presence of mutant MPL has narrow and inconsistent phenotypic effect in PMF and does not influence overall or leukemia-free survival.

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.

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.

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.