We have previously published our observations regarding targeted next generation sequencing (NGS) in primary myelofibrosis (PMF), essential thrombocythemia (ET) and polycythemia vera (PV) (Blood Advances 2016 1:105; Blood Advances 2016 1:21). In the current study, we took a similar approach towards paired-sample NGS analysis in patients with myelofibrosis (MF), including PMF and post-PV/ET MF, and in whom samples were collected both at the time of chronic phase (CP) and blast phase (BP) disease.


Diagnoses of PMF, post-PV MF, post-ET MF and MF-BP were according to WHO criteria (Blood 2016;127:2391). The study population was selected on the basis of the availability of stored DNA, both at the time of CP and BP disease. Targeted capture assays were carried out on bone marrow or whole blood DNA, according to previously published methods, using a panel of 39 genes (Blood Advances 2016 1:105).


i) Clinical information

Paired samples were available in 18 consecutive patients (67% males): 13 PMF and 5 post-PV/ET MF. Clinical data at time of CP (BP) disease included median age 62 years (65 years), transfusion need in 17% (44%), median hemoglobin 10.4 g/dL (9 g/dL), median platelet count 254 x 10(9)/L (69 x 10(9)/L) and median leukocyte count 10.4 x 10(9)/L (22.8 x 10(9)/L). Median (range) of circulating blasts was 0 (0-3) for CP and 31 (1-89) for BP disease. Karyotype was abnormal in 44% (17% unfavorable) and 87% (69% unfavorable) of patients with CP and BP disease, respectively.

ii) Driver mutation distribution in CP vs BP disease

JAK2, CALR and MPL mutations were detected in 12 (67%), 4 (22%) and 1 (6%) patients at time of CP disease; one JAK2 -mutated patient also expressed a CALR mutation. At the time of leukemic transformation, the driver mutational distribution was similar with the exception of one patient who became JAK2 -unmutated.

iii) Mutations that were not acquired at time of leukemic transformation: SRSF2, IDH2, SETBP1, JAK3, U2AF1 and DNMT3B

Mutations seen during CP disease and were not acquired at the time of leukemic transformation included SRSF2 (n=5), IDH2 (n=2), U2AF1 (n=2), and one each of SETBP1, JAK3, and DNMT3B . Among these, one of the U2AF1 and the DNMT3B mutations were no longer detected at time of leukemic transformation. Cytogenetic clonal evolution was demonstrated in 4 of the 5 patients with SRSF2 mutations, with acquisition of new, mostly unfavorable, cytogenetic abnormalities.

iv) Mutations seen only at time of leukemic transformation: FLT3, TP53, KIT, JAK1, SF3B1, BCOR, IDH1 and SUZ12

FLT3 (n=4), TP53 (n=2), KIT (n=2) and one each of JAK1, SF3B, BCOR, IDH1 and SUZ12 mutations were seen only at the time of leukemic transformation. One of the FLT3 -mutated patients also expressed TP53 . Karyotype during CP disease was normal in all 5 patients with acquired FLT3 and/or TP53 mutations and evolved into unfavorable karyotype in 4 of the 5 patients, at the time of leukemic transformation; karyotype remained normal in the one patient with concurrent FLT3 and TP53 mutations. None of the acquired cytogenetic abnormalities in TP53 -mutated patients involved chromosome 17p13.1.

v) Mutations with increased frequency at time of leukemic transformation: ASXL1, EZH2, RUNX1, SH2B3, TET2, PTPN11, NRAS, DNMT3A

ASXL1 (n=9; 5 seen in CP/BP and 4 in BP only), EZH2 (n=5; 4 seen in BP only and 1 in CP/BP), RUNX1 (n=4; 3 seen in BP only and 1 in CP/BP), SH2B3 (n=4; 3 seen in BP only and 1 in CP/BP), TET2 (n=3; 2 seen in BP only and 1 in CP/BP), PTPN11 (n=3; 2 seen in BP only and 1 in CP/BP), NRAS (n=3; one each seen in CP, BP and CP/BP) and DNMT3A (n=3; 2 seen in CP/BP and 1 in BP only). Among the 10 ASXL1 -mutated patients at time of BP disease, 4 also expressed EZH2, 3 SH2B3, 2 TET2, and 1 each DNMT3A, RUNX1, PTPN11 and NRAS . Also in these ASXL1 -mutated patients, karyotype during CP disease was normal in all 7 evaluable cases and remained normal in 2 cases during BP disease; the remaining evolved into unfavorable karyotype.


The current study identifies TP53 and FLT3 as recurrent and BP-specific mutations in MF. The study also demonstrates frequent acquisition of ASXL1, EZH2, RUNX1 and SH2B3 mutations during leukemic transformation. These observations, combined with the demonstration of incomplete concordance between molecular and cytogenetic clonal evolution, suggest pathogenetic contribution from both the listed mutations and other genetic alterations hidden within gross chromosomal abnormalities.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.