Hui Hao-Shen

Clonal evolution and clinical correlates of somatic mutations in myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are a group of clonal disorders characterized by aberrant hematopoietic proliferation and an increased tendency toward leukemic transformation. We used targeted next-generation sequencing (NGS) of 104 genes to detect somatic mutations in a cohort of 197 MPN patients and followed clonal evolution and the impact on clinical outcome. Mutations in calreticulin (CALR) were detected using a sensitive allele-specific polymerase chain reaction. We observed somatic mutations in 90% of patients, and 37% carried somatic mutations other than JAK2 V617F and CALR. The presence of 2 or more somatic mutations significantly reduced overall survival and increased the risk of transformation into acute myeloid leukemia. In particular, somatic mutations with loss of heterozygosity in TP53 were strongly associated with leukemic transformation. We used NGS to follow and quantitate somatic mutations in serial samples from MPN patients. Surprisingly, the number of mutations between early and late patient samples did not significantly change, and during a total follow-up of 133 patient years, only 2 new mutations appeared, suggesting that the mutation rate in MPN is rather low. Our data show that comprehensive mutational screening at diagnosis and during follow-up has considerable potential to identify patients at high risk of disease progression.

Clonal analysis of TET2 and JAK2 mutations suggests that TET2 can be a late event in the progression of myeloproliferative neoplasms.

Somatic mutations in TET2 occur in patients with myeloproliferative neoplasms and other hematologic malignancies. It has been suggested that TET2 is a tumor suppressor gene and mutations in TET2 precede the acquisition of JAK2-V617F. To examine the order of events, we performed colony assays and genotyped TET2 and JAK2 in individual colonies. In 4 of 8 myeloproliferative neoplasm patients, we found that some colonies with mutated TET2 carried wild-type JAK2, whereas others were JAK2-V617F positive, indicating that TET2 occurred before JAK2-V617F. One of these patients carried a germline TET2 mutation. However, in 2 other patients, we obtained data compatible with the opposite order of events, with JAK2 exon 12 mutation preceding TET2 mutation in one case. Finally, in 2 of 8 patients, the TET2 and JAK2-V617F mutations defined 2 separate clones. The lack of a strict temporal order of occurrence makes it unlikely that mutations in TET2 represent a predisposing event for acquiring mutations in JAK2.

Clonal analysis of deletions on chromosome 20q and JAK2-V617F in MPD suggests that del20q acts independently and is not one of the predisposing mutations for JAK2-V617F

We developed a real-time copy number polymerase chain reaction assay for deletions on chromosome 20q (del20q), screened peripheral blood granulocytes from 664 patients with myeloproliferative disorders, and identified 19 patients with del20q (2.9%), of which 14 (74%) were also positive for JAK2-V617F. To examine the temporal relationship between the occurrence of del20q and JAK2-V617F, we performed colony assays in methylcellulose, picked individual burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte (CFU-G) colonies, and genotyped each colony individually for del20q and JAK2-V617F. In 2 of 9 patients, we found that some colonies with del20q carried only wild-type JAK2, whereas other del20q colonies were JAK2-V617F positive, indicating that del20q occurred before the acquisition of JAK2-V617F. However, in colonies from 3 of 9 patients, we observed the opposite order of events. The lack of a strict temporal order of occurrence makes it doubtful that del20q represents a predisposing event for JAK2-V617F. In 2 patients with JAK2-V617F and 1 patient with MPL-W515L, microsatellite analysis revealed that del20q affected chromosomes of different parental origin and/or 9pLOH occurred at least twice. The fact that rare somatic events, such as del20q or 9pLOH, occurred more than once in subclones from the same patients suggests that the myeloproliferative disorder clone carries a predisposition to acquiring such genetic alterations.

Pronounced thrombocytosis in transgenic mice expressing reduced levels of Mpl in platelets and terminally differentiated megakaryocytes

We generated mice expressing a full-length Mpl transgene under the control of a 2-kb Mpl promoter in an Mpl(-/-) background, effectively obtaining mice that express full-length Mpl in the absence of other Mpl isoforms. These mice developed thrombocytosis with platelet levels approximately 5-fold higher than wild-type controls and markedly increased megakaryocyte numbers. The reintroduction of one wild-type Mpl allele restored normal platelet counts. We excluded the deletion of Mpl-tr, a dominant-negative isoform, as the underlying molecular cause for thrombocytosis. Instead, we found that transgene expression driven by the 2-kb Mpl promoter fragment was decreased during late megakaryocyte maturation, resulting in strongly diminished Mpl protein expression in platelets. Because platelets exert a negative feedback on thrombopoiesis by binding and consuming Tpo in the circulation through Mpl, we propose that the severe reduction of Mpl protein in platelets in Mpl-transgenic Mpl(-/-) mice shifts the equilibrium of this feedback loop, resulting in markedly elevated levels of megakaryocytes and platelets at steady state. Although the mechanism causing decreased expression of Mpl protein in platelets from patients with myeloproliferative disorders differs from this transgenic model, our results suggest that lowering Mpl protein in platelets could contribute to raising the platelet count.

The allele burden of JAK2 mutations remains stable over several years in patients with myeloproliferative disorders

This study shows that mutant allele burden remains stable overall several years in patients with JAK2 (V617F)-positive myeloproliferative disorders. In a retrospective single center study we determined the time course of the JAK2-V617F or JAK2 exon 12 allele burden in DNA from purified granulocytes from 48 patients with myeloproliferative disorders. The percentage of change between the first and last sample in JAK2-V617F positive patients without cytoreductive therapy (n=16) was only +9% during a follow-up of 36±13 months, reflecting a remarkably stable mutant allele burden. When treatment with hydroxyurea was initiated during the course of the study, we observed a significant decrease of the JAK2-V617F allele burden (n=6). However, in JAK2-V617F positive patients who were already on hydroxyurea treatment before the first blood sampling (n=14), we observed stable allelic ratios with a variance of only +3% during a follow-up of 34±16 months. Our data suggest that in untreated myeloproliferative disorders patients, from whom samples at diagnosis are not available, the JAK2 allele burden determined at later stages could be equally informative.

Differential effects of hydroxyurea and INC424 on mutant allele burden and myeloproliferative phenotype in a JAK2-V617F polycythemia vera mouse model

To establish a preclinical animal model for testing drugs with potential effects on myeloproliferative neoplasms (MPNs), we first performed a detailed phenotypic characterization of Cre-inducible transgenic JAK2-V617F mice. Deleting the conditional mouse Jak2-knockout alleles increased erythropoiesis and accentuated the polycythemia vera phenotype, but did not alter platelet or granulocyte levels. In a transplantation assay, JAK2-V617F(+) BM cells had an advantage over wild-type competitor cells. Using this competitive repopulation assay, we compared the effects of INC424 (ruxolitinib), a dual Jak1/Jak2 inhibitor, and hydroxyurea (HU). HU led to weight loss, but did not reduce spleen weight. The hematologic parameters were lowered and a slight decrease of the mutant allele burden was noted. INC424 had little effect on body weight, but strongly decreased spleen size and rapidly normalized RBC and neutrophil parameters. No significant decrease in the mutant allele burden was observed. INC424 reduced the phospho-Stat5 levels, whereas HU strongly increased phospho-Stat5, most likely because of the elevated erythropoietin levels in response to the HU-induced anemia. This compensatory increase in JAK/STAT signaling may counteract the beneficial effects of cytoreduction at higher doses of HU and represents an adverse effect that should be avoided.

Loss of Stat1 decreases megakaryopoiesis and favors erythropoiesis in a JAK2 -V617F–driven mouse model of MPNs

The interferon-γ (IFNγ)/signal transducer and activator of transcription 1 (Stat1) pathway shows higher activity in patients with essential thrombocythemia (ET) than in polycythemia vera (PV) and was proposed to be promoting the ET phenotype. We explored the phenotypic consequences of Stat1 deficiency on the effects of Janus kinase 2 (JAK2)-V617F in vivo by crossing mice expressing JAK2-V617F with Stat1 knockout mice. JAK2-V617F;Stat1(-/-) double transgenic mice showed higher red cell parameters and lower platelet counts compared with JAK2-V617F;Stat1(+/+) mice. Bone marrow transplantation reproduced these phenotypic changes in wild-type recipients, demonstrating that the effect of Stat1 is cell-intrinsic and does not require a Stat1-deficient microenvironment. Deletion of Stat1 increased burst-forming unit-erythroid and reduced colony-forming unit-megakaryocyte colony formation driven by JAK2-V617F, but was not sufficient to completely normalize the platelet count. Gata1, a key regulator of megakaryopoiesis and erythropoiesis, was decreased in Stat1-deficient platelets. V617F transgenic mice with thrombocytosis had higher serum levels of IFNγ than normal controls and patients with ET showed higher IFNγ serum levels than patients with PV. Together, these results support the concept that activating Stat1 in the presence of JAK2-V617F, for example, through IFNγ, constrains erythroid differentiation and promotes megakaryocytic development, resulting in ET phenotype.

Deletion of Stat3 in hematopoietic cells enhances thrombocytosis and shortens survival in a JAK2-V617F mouse model of MPN

The acquired somatic JAK2-V617F mutation is present in >80% of patients with myeloproliferative neoplasms (MPNs). Stat3 plays a role in hematopoietic homeostasis and might influence the JAK2-V617F-driven MPN phenotype. We crossed our transgenic SclCre;V617F mice with a conditional Stat3 knockout strain and performed bone marrow transplantations into lethally irradiated recipient mice. The deletion of Stat3 increased the platelet numbers in SclCre;V617F;Stat3(fl/fl) mice compared with SclCre;V617F;Stat3(fl/+) or SclCre;V617F;Stat3(+/+) mice. Stat3 deletion also normalized JAK2-V617F-induced neutrophilia. Megakaryocyte progenitors were elevated, especially in the spleen, and a slight increase in myelofibrosis was noted. We observed increased mRNA expression levels of Stat1 and Stat1 target genes and augmented phosphorylation of Stat1 protein in bone marrow and spleen of JAK2-V617F mice after Stat3 deletion. The survival of Stat3-deficient mice expressing JAK2-V617F was reduced. Inflammatory bowel disease, previously associated with shortened survival of Stat3-deficient mice, was less prominent in the bone marrow transplantation setting, possibly by limiting deletion of Stat3 to hematopoietic tissues only. In conclusion, deletion of Stat3 in hematopoietic cells from JAK2-V617F mice did not ameliorate the course of MPN, but rather enhanced thrombocytosis and shortened the overall survival.

JAK2 exon 12 mutant mice display isolated erythrocytosis and changes in iron metabolism favoring increased erythropoiesis

Mutations in JAK2 exon 12 are frequently found in patients with polycythemia vera (PV) that do not carry a JAK2-V617F mutation. The majority of these patients display isolated erythrocytosis. We generated a mouse model that expresses JAK2-N542-E543del, the most frequent JAK2 exon 12 mutation found in PV patients. Mice expressing the human JAK2-N542-E543del (Ex12) showed a strong increase in red blood cell parameters but normal neutrophil and platelet counts, and reduced overall survival. Erythropoiesis was increased in the bone marrow and spleen, with normal megakaryopoiesis and absence of myelofibrosis in histopathology. Erythroid progenitors and precursors were increased in hematopoietic tissues, but the numbers of megakaryocytic precursors were unchanged. Phosphorylation Stat3 and Erk1/2 proteins were increased, and a trend toward increased phospho-Stat5 and phospho-Stat1 was noted. However, Stat1 knock out in Ex12 mice induced no changes in platelet or red cell parameters, indicating that Stat1 does not play a central role in mediating the effects of Ex12 signaling on megakaryopoiesis or erythropoiesis. Ex12 mice showed decreased expression of hepcidin and increased expression of transferrin receptor-1 and erythroferrone, suggesting that the strong erythroid phenotype in Ex12 mutant mice is favored by changes in iron metabolism that optimize iron availability to allow maximal production of red cells.

Complex subclone structure that responds differentially to therapy in a patient with essential thrombocythemia and chronic myeloid leukemia

To the editor: BCR-ABL translocation and JAK2-V617F mutation can sometimes be found concomitantly in the same patient (reviewed in Hummel et al[1][1]). To date, 4 studies have examined the chronology and architecture of BCR-ABL and JAK2-V617F clones at the molecular level. In 3 studies, the 2