Christy Finke

TET2 mutations and their clinical correlates in polycythemia vera, essential thrombocythemia and myelofibrosis

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 ∼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 ∼16, 5, 17, 14, 14 and 17% (P=0.50). Mutant TET2 was detected in ∼17 and ∼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 ∼23% in patients ⩾60 years old versus ∼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.

Mutations and prognosis in primary myelofibrosis

Patient outcome in primary myelofibrosis (PMF) is significantly influenced by karyotype. We studied 879 PMF patients to determine the individual and combinatorial prognostic relevance of somatic mutations. Analysis was performed in 483 European patients and the seminal observations were validated in 396 Mayo Clinic patients. Samples from the European cohort, collected at time of diagnosis, were analyzed for mutations in ASXL1, SRSF2, EZH2, TET2, DNMT3A, CBL, IDH1, IDH2, MPL and JAK2. Of these, ASXL1, SRSF2 and EZH2 mutations inter-independently predicted shortened survival. However, only ASXL1 mutations (HR: 2.02; P<0.001) remained significant in the context of the International Prognostic Scoring System (IPSS). These observations were validated in the Mayo Clinic cohort where mutation and survival analyses were performed from time of referral. ASXL1, SRSF2 and EZH2 mutations were independently associated with poor survival, but only ASXL1 mutations held their prognostic relevance (HR: 1.4; P=0.04) independent of the Dynamic IPSS (DIPSS)-plus model, which incorporates cytogenetic risk. In the European cohort, leukemia-free survival was negatively affected by IDH1/2, SRSF2 and ASXL1 mutations and in the Mayo cohort by IDH1 and SRSF2 mutations. Mutational profiling for ASXL1, EZH2, SRSF2 and IDH identifies PMF patients who are at risk for premature death or leukemic transformation.

Systemic mastocytosis in 342 consecutive adults: survival studies and prognostic factors

Clinical phenotype in systemic mastocytosis (SM) is markedly variable, which complicates prognostication and decision making regarding the choice and timing of therapy. In a retrospective study of 342 consecutive adult patients with SM seen at the Mayo Clinic between 1976 and 2007, disease subdesignation according to the World Health Organization (WHO) proposal was indolent (ISM) in 159 (46%), with associated clonal hematologic non-mast cell lineage disease (SM-AHNMD) in 138 (40%), aggressive (ASM) in 41 (12%), and mast cell leukemia in 4 (1%). KITD816V was detected in bone marrow-derived DNA by allele-specific polymerase chain reaction (PCR) in 68% of 165 patients evaluated (ISM, 78%; ASM, 82%; SM-AHNMD, 60%; P = .03); JAK2V617F was detected in 4%, all in SM-AHNMD. Compared with those with nonindolent SM, life expectancy in ISM was superior and not significantly different from that of the age- and sex-matched US population. In addition, multivariable analysis identified advanced age, weight loss, anemia, thrombocytopenia, hypoalbuminemia, and excess bone marrow blasts as independent adverse prognostic factors for survival. The current study validates the prognostic relevance of the WHO subclassification of SM and provides additional information of value in terms of both risk stratification and interpretation of clinical presentation and laboratory results.

Circulating interleukin (IL)-8, IL-2R, IL-12, and IL-15 levels are independently prognostic in primary myelofibrosis: A comprehensive cytokine profiling study

PURPOSE Abnormal cytokine expression accompanies myelofibrosis and might be a therapeutic target for Janus-associated kinase (JAK) inhibitor drugs. This study describes the spectrum of plasma cytokine abnormalities in primary myelofibrosis (PMF) and examines their phenotypic correlates and prognostic significance. PATIENTS AND METHODS Patients included in this study were required to have archived plasma, bone marrow biopsy, and cytogenetic information available at the time of first referral to the Mayo Clinic. Multiplex biometric sandwich immunoassay was used to measure plasma levels of 30 cytokines. RESULTS In total, 127 PMF patients were studied; comparison with normal controls (n = 35) revealed significantly increased interleukin-1β (IL-1β), IL-1RA, IL-2R, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, tumor necrosis factor α (TNF-α), granulocyte colony-stimulating factor (G-CSF), interferon alfa (IFN-α), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, hepatocyte growth factor (HGF), IFN-γ-inducible protein 10 (IP-10), monokine induced by IFN-γ (MIG), monocyte chemotactic protein 1 (MCP-1), and vascular endothelial growth factor (VEGF) levels and decreased IFN-γ levels. In treatment-naive patients (n = 90), increased levels of IL-8 (P < .001), IL-2R (P < .001), IL-12 (P < .001), IL-15 (P = .001), and IP-10 (P = .003) were independently predictive of inferior survival. A similar multivariable analysis that included all 127 study patients confirmed the prognostic value of these five cytokines, and IL-8, IL-2R, IL-12, and IL-15 remained significant when risk stratification, according to the recently revised Dynamic International Prognostic Scoring System (DIPSS plus), was added to the multivariable model. Leukemia-free survival was predicted by IL-8, which was also the only cytokine associated with ≥ 1% circulating blasts. Other cytokine-phenotype associations included increased IL-8 and constitutional symptoms; IL-2R, IL-12, and transfusion need; IL-2R, IL-8, and leukocytosis; IP-10 and thrombocytopenia; HGF, MIG, IL-1RA, and marked splenomegaly; and IL-1RA, IL-2R, IP-10, MIP-1β, and JAK2V617F. A two-cytokine (IL-8/IL-2R) -based risk categorization delineated prognostically different groups within specific DIPSS plus risk categories. CONCLUSION This study signifies the presence of specific cytokine-phenotype associations in PMF and a prognostically relevant plasma cytokine signature that might prove useful as a laboratory tool for predicting and monitoring treatment response.

CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: Clinical, cytogenetic and molecular comparisons

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.

IDH1 and IDH2 Mutation Studies in 1473 Patients with Chronic-, Fibrotic- or Blast-Phase Essential Thrombocythemia, Polycythemia Vera or Myelofibrosis

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.

TG101209, a small molecule JAK2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated JAK2V617F and MPLW515L/K mutations

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling, which is implicated in MPD pathogenesis. We developed TG101209, an orally bioavailable small molecule that potently inhibits JAK2 (IC50=6 nM), FLT3 (IC50=25 nM) and RET (IC50=17 nM) kinases, with significantly less activity against other tyrosine kinases including JAK3 (IC50=169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC50 of ∼200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209-induced cell cycle arrest and apoptosis, and inhibited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore, TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations.

Prevalence and clinicopathologic correlates of JAK2 exon 12 mutations in JAK2V617F-negative polycythemia vera

After accounting for misdiagnosis and treatment effect, allele-specific (AS)-PCR detects the JAK2V617F mutation in >95% of polycythemia vera (PV) patients. Using database inquiry, we identified 6 of a total 220 cases with PV that were JAK2V617F-negative (prevalence=3%). Of these, five cases (∼80%) were found to harbor one of the two JAK2 exon 12 mutations (F537-K539delinsL or N542-E543del) in bone marrow (BM) and/or peripheral blood cells. Similar screening of six additional cases – three each with idiopathic erythrocytosis (IE) or otherwise unexplained erythrocytosis (UE) – did not reveal either JAK2V617F or JAK2 exon 12 mutations. We found JAK2 exon 12 mutations in PV cases to be readily detected by both DNA sequencing and AS-PCR, regardless of whether BM or peripheral blood cells were used as the source for DNA. Although erythroid hyperplasia was the predominant histologic feature on BM examination, megakaryocyte abnormalities and reticulin fibrosis were noted in most PV patients harboring exon 12 mutations. However, similar BM morphologic changes can also be seen in some JAK2V617F-positive PV cases; therefore, distinct genotype–phenotype association cannot be established.

Low JAK2V617F allele burden in primary myelofibrosis, compared to either a higher allele burden or unmutated status, is associated with inferior overall and leukemia-free survival

The clinical relevance of JAK2V617F allele burden in primary myelofibrosis (PMF) has not been previously studied. Bone marrow-derived DNA from 199 patients with PMF was subjected to qualitative (n=199) and quantitative (n=129) analysis for V617F. Mutational frequency was 58% and median mutant allele burden ratio in V617F-positive patients was 29% (range, 1–74%). Multivariable analysis identified older age, platelet count ⩾100 × 109 l−1 and peripheral blood blast percentage <3% as being associated with a positive mutational status. The mere presence of the mutation did not affect the incidence of thrombosis (P=0.78), overall survival (P=0.22) or leukemia-free survival (P=0.5). The 129 patients with allele burden information were divided into four groups: V617F-negative (n=53) and V617F-positive with mutant allele burden in the lower quartile (n=19), middle quartiles (n=38) or upper quartile (n=19) range. Kaplan–Meier plots revealed significantly shortened overall (P=0.0008) and leukemia-free (P=0.01) survival for the lower quartile, but not for upper quartile allele burden group; independent prognostic relevance was validated by multivariable analysis. We conclude that low V617F allele burden in PMF might indicate the presence of an overriding V617F-negative clone that confers a more aggressive disease phenotype.

Long-term survival and blast transformation in molecularly annotated essential thrombocythemia, polycythemia vera, and myelofibrosis

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.