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Abstract: DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematologic neoplasms. Although individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here, we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity (acute respiratory distress syndrome [ARDS]) and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant (DNMT3Amt) CH on COVID-19-related ARDS and mortality. Using single-cell proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid lineage cells. Using single-cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of interleukin-32 (IL-32), a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single-cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Hence, we demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19-related ARDS and mortality. DNMT3Amt CH in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can thus serve as a potential biomarker for adverse outcomes in COVID-19., (© 2025 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Abstract: This phase 2 trial assessed high-dose IV ascorbic acid in TET2 mutant clonal cytopenia. Eight of 10 patients were eligible for response assessment, with no responses at week 20 by International Working Group Myelodysplasia Syndromes/Neoplasms criteria. This trial was registered at www.clinicaltrials.gov as #NCT03418038., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

The current study was inspired by observations from exploratory analyses of an institutional cohort with chronic myelomonocytic leukemia (CMML; N = 398) that revealed no instances of blast transformation in the seven patients with plant homeodomain finger protein 6 (PHF6) mutation (PHF6 MUT ). A subsequent Mayo Clinic enterprise-wide database search identified 28 more cases with PHF6 MUT . Compared with their wild-type PHF6 counterparts (PHF6 WT ; N = 391), PHF6 MUT cases (N = 35) were more likely to co-express TET2 (89% vs. 45%; p < .01), RUNX1 (29% vs. 14%; p = .03), CBL (14% vs. 2%; p < .01), and U2AF1 (17% vs. 6%; p = .04) and less likely SRSF2 (23% vs. 45%; p < .01) mutation. They were also more likely to display loss of Y chromosome (LoY; 21% vs. 2%; p < .01) and platelets <100 × 10 9 /L (83% vs. 51%; p < .01). Multivariable analysis identified PHF6 MUT (HR 0.28, 95% CI 0.15-0.50) and DNMT3A MUT (HR 5.8, 95% CI 3.3-10.5) as the strongest molecular predictors of overall survival. The same was true for blast transformation-free survival with corresponding HR (95% CI) of 0.08 (0.01-0.6) and 9.5 (3.8-23.5). At median 20 months follow-up, blast transformation was documented in none of the 33 patients with PHF6 MUT /DNMT3A WT but in 6 (32%) of 19 with DNMT3A MUT and 74 (20%) of 374 with PHF6 WT /DNMT3A WT (p < .01). The specific molecular signatures sustained their significant predictive performance in the context of the CMML-specific molecular prognostic model (CPSS-mol). PHF6 MUT identifies a unique subset of patients with CMML characterized by thrombocytopenia, higher prevalence of LoY, and superior prognosis., (© 2024 The Author(s). American Journal of Hematology published by Wiley Periodicals LLC.)

Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1-/- accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1-/- (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations., (© 2022 by The American Society of Hematology.)

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.

We examined the influence of mutations and karyotype on conventional treatment response, specifically hematological improvement in anemia, in primary myelodysplastic syndromes (MDS). Cytogenetic and next generation sequencing (NGS)-derived mutation information was available in 357 patients (median age 74 years; 70% males); the revised international prognostic scoring system risk distribution was very high in 11%, high 15%, intermediate 17%, low 40% and very low 16%. At least one mutation was detected in 81% of patients; most frequent were SF3B1 (32%), ASXL1 (27%), TET2 (24%) and U2AF1 (15%). At median follow-up of 24 months, treatment with hypomethylating agents (HMAs) was documented in 121 (34%) patients, lenalidomide (LEN) in 55 (15%), and erythropoiesis stimulating agents (ESAs) in 136 (38%). ASXL1 mutations adversely affected response to HMAs (27% vs 48%; P = 0.02) and LEN (9% vs 43%; P = 0.04), but not ESAs (P = 0.6). LEN response was also adversely affected by U2AF1 mutations (0% vs 42%; P = 0.02) and high risk karyotype (0% vs 41% in intermediate vs 47% in low risk; P = 0.01). Patients with SF3B1 mutations were more likely to respond to LEN (56% vs 27%; P = 0.04). Contrary to previous reports, we found no association between TET2 mutations and HMA treatment response (40% vs 41%; P = 0.9), even in the absence of ASXL1 mutations (P = 0.4).We conclude that ASXL1 mutations in MDS predict inferior response to treatment with both HMAs and LEN; response to LEN was also compromised by U2AF1 mutations and high risk karyotype; SF3B1 mutations identified patients likely to respond to LEN., (© 2018 Wiley Periodicals, Inc.)

To develop a genetic risk model for primary myelodysplastic syndromes (MDS), we queried the prognostic significance of next-generation sequencing (NGS)-derived mutations, in the context of the Mayo cytogenetic risk stratification, which includes high-risk (monosomal karyotype; MK), intermediate-risk (non-MK, classified as intermediate/poor/very poor, per the revised international prognostic scoring system; IPSS-R), and low-risk (classified as good/very good, per IPSS-R). Univariate analysis in 300 consecutive patients with primary MDS identified TP53, RUNX1, U2AF1, ASXL1, EZH2, and SRSF2 mutations as "unfavorable" and SF3B1 as "favorable" risk factors for survival; for the purposes of the current study, the absence of SF3B1 mutation was accordingly dubbed as an "adverse" mutation. Analysis adjusted for age and MK, based on our previous observation of significant clustering between MK and TP53 mutations, confirmed independent prognostic contribution from RUNX1, ASXL1, and SF3B1 mutations. Multivariable analysis that included age, the Mayo cytogenetics risk model and the number of adverse mutations resulted in HRs (95% CI) of 5.3 (2.5-10.3) for presence of three adverse mutations, 2.4 (1.6-3.7) for presence of two adverse mutations, 1.5 (1.02-2.2) for presence of one adverse mutation, 5.6 (3.4-9.1) for high-risk karyotype, 1.5 (1.1-2.2) for intermediate-risk karyotype and 2.4 (1.8-3.3) for age >70 years; HR-weighted risk point assignment generated a three-tiered genetic risk model: high (N = 65; 5-year survival 2%), intermediate (N = 100; 5-year survival 18%), and low (N = 135; 5-year survival 56%). The current study provides a practically simple risk model in MDS that is based on age, karyotype, and mutations only., (© 2018 Wiley Periodicals, Inc.)

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.)

A 27-gene panel was used for next-generation sequencing (NGS) in 179 patients (median age 73 years) with primary myelodysplastic syndromes (MDS); risk distribution according to the revised International Prognostic Scoring System (IPSS-R) was 11% very high, 18% high, 17% intermediate, 38% low and 16% very low. At least one mutation/variant was detected in 147 (82%) patients; 23% harbored three or more mutations/variants. The most frequent mutations/variants included ASXL1 (30%), TET2 (25%), SF3B1 (20%), U2AF1 (16%), SRSF2 (16%), TP53 (13%), RUNX1 (11%), and DNMT3A (10%). At a median follow up of 30 months, 148 (83%) deaths and 26 (15%) leukemic transformations were recorded. Multivariable analysis of mutations/variants identified ASXL1 (HR 1.7, 95% CI 1.2-2.5), SETBP1 (HR 4.1, 95% CI 1.6-10.2) and TP53 (HR 2.2, 95% CI 1.3-3.4) as risk factors for overall and SRSF2 (HR 3.9, 95% CI 1.5-10.2), IDH2 (HR 3.7, 95% CI 1.2-11.4), and CSF3R (HR 6.0, 95% CI 1.6-22.6) for leukemia-free survival. Addition of age to the multivariable model did not affect these results while accounting for IPSS-R weakened the significance of TP53 mutations/variants (P = .1). An apparently favorable survival impact of SF3B1 mutations was no longer evident after adjustment for IPSS-R. Approximately 41% and 20% of patients harbored at least one adverse mutation/variant for overall and leukemia-free survival, respectively. Number of mutations/variants did not provide additional prognostic value. The survival impact of adverse mutations was most evident in IPSS-R very low/low risk patients. These observations suggest that targeted NGS might assist in treatment decision-making in lower risk MDS., (© 2017 Wiley Periodicals, Inc.)

DNMT3A mutations are seen in ∼5% of patients with chronic myelomonocytic leukemia (CMML) and thus far, have had an indeterminate prognostic impact on survival. We carried out this study to assess the prognostic impact of DNMT3A mutations on a larger informative cohort of CMML patients (n = 261). DNMT3A mutations were seen in 6% (n = 16); 56% (n = 9) male, with a median age of 64 years. Eighty-one % of DNMT3A mutations were missense, with the Arg882 mutational hot spot accounting for 63% of all changes. Five (31%) patients had an abnormal karyotype whereas concurrent gene mutations (SF3B1/SRSF2/U2AF1-56%, TET2-50%, and ASXL1-25%) were seen in all patients. Apart from a higher frequency of SF3B1 (P = 0.0001) and PTPN11 (P = 0.005) mutations and a lower frequency of SRSF2 (P = 0.004) mutations, there were no significant differences between DNMT3A mutated patients and their wildtype counterparts. In univariate analysis, survival was shorter in DNMT3A mutated (median 8 months) versus wildtype (median 27 months) patients (P = 0.0007; HR 2.9, 95% CI 1.5-5.7); with other variables of significance including lower hemoglobin (P = 0.002), higher leukocyte count (P = 0.0009), higher monocyte count (P = 0.0012), circulating blast % (P = 0.001), circulating immature myeloid cells (P = 0.01), bone marrow blast % (P = 0.045), abnormal karyotype (P = 0.02), and ASXL1 (P = 0.01) mutations. In a multivariable model that included the aforementioned variables, when both DNMT3A and ASXL1 mutations were added, only DNMT3A (P < 0.0001) and ASXL1 (P = 0.004) mutations remained significant. DNMT3A mutations were also predictive of a shortened leukemia-free survival. These findings warrant inclusion of DNMT3A mutations in molecularly integrated CMML prognostic models. Am. J. Hematol. 92:56-61, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

In routine practice, the World Health Organization classification of systemic mastocytosis (SM) is also the de facto prognostic system; a core value is distinguishing indolent (ISM) from advanced SM (includes aggressive SM [ASM], SM with associated hematological neoplasm [SM-AHN] and mast cell leukemia [MCL]). We sequenced 27 genes in 150 SM patients to identify mutations that could be integrated into a clinical-molecular prognostic model for survival. Forty four patients (29%) had ISM, 25 (17%) ASM, 80 (53%) SM-AHN and 1 (0.7%) MCL; overall KITD816V prevalence was 75%. In 87 patients, 148 non-KIT mutations were detected; the most frequently mutated genes were TET2 (29%), ASXL1 (17%), and CBL (11%), with significantly higher mutation frequency in SM-AHN > ASM > ISM (P < 0.0001). In advanced SM, ASXL1 and RUNX1 mutations were associated with inferior survival. In multivariate analysis, age > 60 years (HR = 2.4), hemoglobin < 10 g/dL or transfusion-dependence (HR = 1.7), platelet count < 150 × 10(9) /L (HR = 3.2), serum albumin < 3.5 g/dL (HR = 2.6), and ASXL1 mutation (HR = 2.3) were associated with inferior survival. A mutation-augmented prognostic scoring system (MAPSS) based on these parameters stratified advanced SM patients into high-, intermediate-, and low-risk groups with median survival of 5, 21 and 86 months, respectively (P < 0.0001). These data should optimize risk-stratification and treatment selection for advanced SM patients. Am. J. Hematol. 91:888-893, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

About 85% of patients with essential thrombocythemia (ET) harbor one of three driver mutations: JAK2, calreticulin (CALR), and MPL; the remaining ( ∼15%) are wild type for all three mutations and are referred to as being "triple negative." Furthermore, CALR mutations in ET are structurally classified as type 1/type 1-like or type 2/type 2-like variants. The objective of the current study was to examine the impact of CALR mutation variant stratified driver mutational status on overall (OS), myelofibrosis-free (MFFS), thrombosis-free, and leukemia-free survival (LFS) in ET; 495 patients (median age 58 years; 61% females) with ET were fully annotated for the their driver mutational status: 321 (65%) harbored JAK2, 109 (22%) CALR, and 12 (2%) MPL mutations and 11% were triple-negative. Among the 109 CALR-mutated cases, 52% were classified as type 1/type 1-like and 48% as type 2/type 2-like. In univariate analysis, triple-negative patients displayed the best and MPL mutated the worst OS (P = 0.007); however, the difference in OS was no longer apparent on multivariable analysis that included age and sex as covariates (P = 0.5). LFS was also similar among the different mutational groups (P = 0.6) whereas MFFS was significantly shorter in MPL-mutated patients on both univariate and multivariable analyses (age-adjusted P = 0.02; HR 7.9, 95% CI 2.0-31.5). Also in multivariable analysis that included thrombosis history, age, and cardiovascular risk factors, the presence of JAK2 or MPL mutations was independently associated with higher risk of thrombosis (P = 0.02; HR 1.9, 95% CI 1.1-3.4). In conclusion, driver mutational status in ET does not appear to influence overall or LFS, even after CALR variant stratification. However, the presence of MPL mutations might be associated with a higher risk of fibrotic transformation and the presence of JAK2/MPL mutations with higher risk of thrombosis., (© 2016 Wiley Periodicals, Inc.)

Background: Vascular events in essential thrombocythemia (ET) are associated with advanced age and thrombosis history. Recent information suggests additional effect from the presence of specific mutations., Objectives: To examine the influence of age and thrombosis history on the reported association between mutational status and thrombosis-free survival in ET., Patients and Methods: Analysis was performed using a Mayo Clinic cohort of 300 ET patients, and key findings were reanalyzed by including additional 102 Italian patients., Results: Among 300 Mayo patients with ET (median age 55 yr, 60% females), mutational frequencies were 53% JAK2, 32% CALR, 3% MPL, and 12% JAK2, CALR and MPL wild type. One hundred and six (35%) patients experienced arterial (n = 75) or venous (n = 43) events, before (n = 55) or after (n = 71) diagnosis. In univariate analysis, compared to JAK2-mutated cases, JAK2, CALR and MPL wild type (HR 0.31, 95% CI 0.11-0.86), and CALR-mutated (0.53, 95% CI 0.30-0.92) patients displayed better thrombosis-free survival. JAK2, CALR, and MPL wild type remained significant (P = 0.03; HR 0.32, 95% CI 0.11-0.9) during multivariable analysis that included age (P = 0.01) and thrombosis history (P = 0.0006); a favorable impact from CALR mutations was of borderline significance (P = 0.1; HR 0.62, 95% CI 0.35-1.1), but became significant (P = 0.02) when multivariable analysis including thrombosis history (P = 0.02) was performed on patients younger than 60 yr of age., Conclusions: The favorable impact of mutational status on thrombosis-free survival in ET might be most evident for JAK2, CALR, and MPL wild type patients, whereas the favorable effect from CALR mutations might be confined to young patients., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Four hundred and nine patients with chronic myelomonocytic leukemia (CMML) were included in the international collaborative study (268 (66%) and 141 (34%) from Mayo clinic and French consortium respectively). Thirty percent displayed an abnormal karyotype, including; 72% sole, 16% two, and 11% complex abnormalities. The most common abnormalities included; +8 (23%), -Y (20%), -7/7q-(14%), 20q- (8%), +21 (8%), and der(3q) (8%). Patients with an abnormal karyotype were more likely to be elderly (P = 0.03), be anemic (P = 0.0009), have leukocytosis (P = 0.02) with neutrophilia (P = 0.03), demonstrate increased circulating immature myeloid cells (P = 0.0003), peripheral blood blasts (P < 0.0001), and bone marrow blasts (P < 0.0001). ASXL1 (P = 0.04) and SF3B1 (P = 0.03) mutations clustered with an abnormal karyotype, whereas SRSF2 (P = 0.02) mutations occurred more commonly with a normal karyotype. A step-wise survival analysis resulted in three distinct cytogenetic risk categories: high (complex and monosomal karyotypes), intermediate (all abnormalities not in the high or low risk groups) and low [normal, sole -Y and sole der (3q)] with median survivals of 3 [hazard ratio (HR) = 8.1, 95% confidence interval (CI) = 4.6-14.2], 20 (HR = 1.7, 95% CI = 1.2-2.3) and 41 months, respectively. In multivariable analysis, this particular cytogenetic risk stratification remained significant in the context of the Molecular Mayo Model (P < 0.0001), MD Anderson prognostic model (P < 0.0001), the GFM CMML model (P < 0.0001) and was effective in predicting leukemic transformation (P = 0.004)., (© 2014 Wiley Periodicals, Inc.)

Janus kinase 2 (JAK2) mutations define polycythemia vera (PV). Calreticulin (CALR) and myeloproliferative leukemia virus oncogene (MPL) mutations are specific to JAK2-unmutated essential thrombocythemia (ET) and primary myelofibrosis (PMF). We examined the effect of these mutations on long-term disease outcome. One thousand five hundred eighty-one patients from the Mayo Clinic (n = 826) and Italy (n = 755) were studied. Fifty-eight percent of Mayo patients were followed until death; median survivals were 19.8 years in ET (n = 292), 13.5 PV (n = 267; hazard ratio [HR], 1.8; 95% confidence interval [CI], 1.4-2.2), and 5.9 PMF (n = 267; HR, 4.5; 95% CI, 3.5-5.7). The survival advantage of ET over PV was not affected by JAK2/CALR/MPL mutational status. Survival in ET was inferior to the age- and sex-matched US population (P < .001). In PMF (n = 428), but not in ET (n = 576), survival and blast transformation (BT) were significantly affected by mutational status; outcome was best in CALR-mutated and worst in triple-negative patients: median survival, 16 vs 2.3 years (HR, 5.1; 95% CI, 3.2-8.0) and BT, 6.5% vs 25% (HR, 7.6; 95% CI, 2.8-20.2), respectively. We conclude that life expectancy in morphologically defined ET is significantly reduced but remains superior to that of PV, regardless of mutational status. In PMF, JAK2/CALR/MPL mutational status is prognostically informative., (© 2014 by The American Society of Hematology.)

CALR (calreticulin) trails JAK2 as the second most mutated gene in essential thrombocythemia (ET). Mutant CALR in ET is a result of frameshift mutations, caused by exon 9 deletions or insertions; type-1, 52-bp deletion (p.L367fs*46), and type-2, 5-bp TTGTC insertion (p.K385fs*47) variants constitute more than 80% of these mutations. The current study includes a total of 1027 patients divided into test (n = 402) and validation (n = 625) cohorts. Among the 402 ET patients in the test cohort, 227 (57%) harbored JAK2, 11 (3%) Myeloproliferative leukemia virus oncogene (MPL), and 114 (28%) CALR mutations; 12% were wild-type for all three mutations (i.e., triple-negative). Among the 114 patients with CALR mutations, 51 (45%) displayed type-1 and 44 (39%) type-2 variants; compared to mutant JAK2, both variants were associated with higher platelet and lower hemoglobin and leukocyte counts. However, male sex was associated with only type-1 (P = 0.005) and younger age with type-2 (P = 0.001) variants. Notably, platelet count was significantly higher in type-2 vs. type-1 CALR-mutated patients (P = 0.03) and the particular observation was validated in the validation cohort that included 111 CALR-mutated ET patients (P = 0.002). These findings, coupled with the recent demonstration of preferential expression of mutant and wild-type CALR in megakaryocytes, suggest differential effects of CALR variants on thrombopoiesis., (© 2014 Wiley Periodicals, Inc.)

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.

A total of 107 patients with chronic-phase primary myelofibrosis (PMF) were screened for TP53 mutations, which were detected in 4 (4%) cases: (i) E204E; GAG>GAA (silent exon 6); (ii) G245D; GGC>GAC (exon 7); (iii) R175H; CGC>CAC (exon 5); and (iv) six base insert (GGCGAG) after bp13767 (exon 6). Three (75%) of the four TP53-mutated cases also carried JAK2V617F whereas none were positive for MPL or IDH mutations. Two of the four TP53 mutated cases were also screened for TET2, ASXL1, DNMT3A, and EZH2 mutations and were negative. There was no significant difference in presenting features or survival between TP53 mutated and unmutated cases. TP53 exon 4 single nucleotide polymporphism (SNPs) data for codon 72 were available on 104 patients and included 56% with homozygous Arg72Arg, 33% with heterozygous Pro72Arg, and 11% with homozygous Pro72Pro. There were no significant differences among the three codon 72 genotypes in terms of presenting characteristics or survival.

Comparative genomic hybridization (CGH), using oligo arrays with either 44,000 or 105,000 oligonucleotides, was performed on granulocyte-derived DNA from 71 patients with BCR-ABL-negative classic myeloproliferative neoplasms (MPNs): 32 primary myelofibrosis (PMF), 26 polycythemia vera (PV) and 13 essential thrombocythemia (ET). Copy number changes (CNCs) were detected in 44%, 35%, and 15% of the cases with PMF, PV and ET, respectively. In ET and PMF, CNCs were more frequently detected in the presence of JAK2V617F (50% vs. 19%; p=0.05). Conventional chromosome analysis was obtained in 57 patients either at diagnosis or within 1 year of the array CGH study; all 21 patients with PV and 11 with ET displayed normal cytogenetic findings despite the presence of CNCs in 29% and 18%, respectively. In PMF, the respective rates of CNCs and abnormal karyotype were 48% and 36%; karyotypic abnormalities, including unbalanced translocations, were often detected by array CGH as chromosomal gains or losses. This preliminary report suggests a potential value for array CGH in terms of both clinical diagnostics and genomic research in MPNs.

Summary: Survival prediction in essential thrombocythaemia (ET) and polycythaemia vera (PV) is currently based on clinically‐derived variables; we examined the possibility of integrating genetic information for predicting survival. To this end, 906 molecularly‐annotated patients (416 Mayo Clinic; 490 University of Florence, Italy), including 502 ET and 404 PV, were recruited. Multivariable analysis identified spliceosome mutations to adversely affect overall (SF3B1, SRSF2 in ET and SRSF2 in PV) and myelofibrosis‐free (U2AF1, SF3B1 in ET) survival; TP53 mutations predicted leukaemic transformation in ET; "adverse" mutations occurred in 51 (10%) ET and 8 (2%) PV patients. We confirmed the independent survival effect of adverse mutations [hazard ratio (HR) 2·4, 95% CI 1·6–3·5], age >60 years (6·6, 4·6–9·7), male sex (1·8, 1·3–2·4) and leukocytosis ≥11 × 109/l (1·6, 1·1–2·2), in ET, and adverse mutations (7·8, 3·1–17·0), age >67 years (5·4, 3·6–8·1), leukocytosis ≥15 × 109/l (2·8, 1·8–4·2) and thrombosis history (2·0, 1·4–2·9), in PV. HR‐based risk point allocation allowed development of three‐tiered mutation‐enhanced international prognostic systems (MIPSS) which were validated in both cohorts and performance was shown to be superior to conventional scoring systems. Spliceosome mutations enhance survival prediction in ET and PV and identify patients at risk for fibrotic progression. TP53 mutations predict leukaemic transformation in ET. [ABSTRACT FROM AUTHOR]