A Pardanani

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.

IDH1 and IDH2 mutation analysis in chronic-and blast-phase myeloproliferative neoplasms

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 ∼21% (7 of 34) for blast-phase MPN and ∼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.

Lenalidomide therapy in del(5)(q31)-associated myelofibrosis: cytogenetic and JAK2V617F molecular remissions

Lenalidomide therapy in del(5)(q31)-associated myelofibrosis: cytogenetic and JAK2 V617F molecular remissions

IDH mutations in primary myelofibrosis predict leukemic transformation and shortened survival: clinical evidence for leukemogenic collaboration with JAK2V617F.

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.

Mayo prognostic model for WHO-defined chronic myelomonocytic leukemia: ASXL1 and spliceosome component mutations and outcomes

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 × 109/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 × 109/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.

A phase-2 trial of low-dose pomalidomide in myelofibrosis.

In a previous study, we reported on the safety and efficacy of low-dose (0.5 mg) pomalidomide and prednisone and pomalidomide alone (2 mg/day), for the treatment of anemia associated with myelofibrosis (MF). The current study examined the value of low-dose pomalidomide alone. The main eligibility criterion was transfusion-dependency or hemoglobin <10 gm per 100 ml. Anemia response was assessed by International Working Group criteria. Pomalidomide (0.5 mg/day) was given to 58 patients (median age 68 years); 46 (79%) were transfusion-dependent and 42 were JAK2V617F positive. Anemia response was documented only in the presence of JAK2V617F (24 vs 0%; P=0.03) but was not further affected by mutant allele burden (P=0.39); 9 of the 10 anemia responders became transfusion independent. Anemia response in JAK2V617F-positive patients was predicted by the presence of pomalidomide-induced basophilia in the first month of therapy (38 vs 6%; P=0.02) or absence of marked splenomegaly (38 vs 11%; P=0.05). A total of 14 (58%) of 24 patients with a platelet count of ⩽100 × 109 cells/l experienced a >50% increment in platelet count. There were no spleen responses. Grade 3 or 4 thrombocytopenia/neutropenia occurred in 2%/0% of patients. Low-dose pomalidomide is effective in the treatment of anemia associated with JAK2V617F-positive MF; response is predicted by early drug-induced basophilia.

Type 1 vs type 2 calreticulin mutations in primary myelofibrosis: Differences in phenotype and prognostic impact

Type 1 vs type 2 calreticulin mutations in primary myelofibrosis: differences in phenotype and prognostic impact

Differential prognostic effect of IDH1 versus IDH2 mutations in myelodysplastic syndromes: a Mayo Clinic Study of 277 patients

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.

JAK2 germline genetic variation affects disease susceptibility in primary myelofibrosis regardless of V617F mutational status: Nullizygosity for the JAK2 46/1 haplotype is associated with inferior survival

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 (∼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.