Effect of mutation order on myeloproliferative neoplasms

Abstract

Background: Cancers result from the accumulation of somatic mutations, and their properties are thought to reflect the sum of these mutations. However, little is known about the effect of the order in which mutations are acquired.

Methods: We determined mutation order in patients with myeloproliferative neoplasms by genotyping hematopoietic colonies or by means of next-generation sequencing. Stem cells and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells.

Results: The age at which a patient presented with a myeloproliferative neoplasm, acquisition of JAK2 V617F homozygosity, and the balance of immature progenitors were all influenced by mutation order. As compared with patients in whom the TET2 mutation was acquired first (hereafter referred to as "TET2-first patients"), patients in whom the Janus kinase 2 (JAK2) mutation was acquired first ("JAK2-first patients") had a greater likelihood of presenting with polycythemia vera than with essential thrombocythemia, an increased risk of thrombosis, and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. Mutation order influenced the proliferative response to JAK2 V617F and the capacity of double-mutant hematopoietic cells and progenitor cells to generate colony-forming cells. Moreover, the hematopoietic stem-and-progenitor-cell compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2-TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevented JAK2 V617F from up-regulating genes associated with proliferation.

Conclusions: The order in which JAK2 and TET2 mutations were acquired influenced clinical features, the response to targeted therapy, the biology of stem and progenitor cells, and clonal evolution in patients with myeloproliferative neoplasms. (Funded by Leukemia and Lymphoma Research and others.).

Figure 1. JAK2 V617F Preceding or Following TET2 Mutations in Patients with Chronic-Phase Myeloproliferative Neoplasms

As shown in Panel A, colonies of erythroid burst-forming units (BFU-E) were grown in a semisolid medium from peripheral-blood mononuclear cells obtained from patients with myeloproliferative neoplasms who carried mutations in TET2 and JAK2. Colonies (20 to 200 per patient, >7000 total) were picked and individually sequenced to determine the clonal composition and order in which mutations were acquired. In Panels B and D, the letters and numbers at the top of each column are patient-identification numbers for individual patients. Numbers beside the patient-identification numbers indicate the total number of colonies in that patient genotyped, with the percentages of total colonies of each genotype shown below. The circles beside these percentages are proportional to the percentages. In each of these 3 patients with myeloproliferative neoplasms who had long-standing disease, the time from diagnosis was 61 months (in a patient with essential thrombocythemia [ET], left column), 67 months (in a patient with polycythemia vera [PV], middle column), and 16 months (in a patient with myelofibrosis [MF], right column). A similar level of clonal heterogeneity was present in the majority of 24 patients with myeloproliferative neoplasms (see Fig. 1 in Supplementary Appendix 1). The mean total duration of disease was 80 months in patients with polycythemia vera, 97 months in patients with essential thrombocythemia, and 85 months in patients with myelofibrosis. J denotes JAK2 V617F mutation, JJ JAK2 V617F homozygosity, NM nonmutated, and T TET2 mutation. In Panel C, the stacked column plot indicates the mutational status of colonies in patients with polycythemia vera, essential thrombocythemia, and myelofibrosis. The total number of colonies per patient is shown at the top of each column; average percentages are shown in cases of repeated assays. Heterozygous and homozygous acquisitions of mutations were each counted as separate mutational events. Subclones containing only the first mutation were more common in patients with polycythemia vera (P = 0.01 by the Mann–Whitney test) and essential thrombocythemia (P = 0.02 by the Mann–Whitney test) than in patients with myelofibrosis. The clonal composition was determined in 12 patients with myeloproliferative neoplasms at intervals of 12 to 44 months; 2 representative patients are shown in Panel D. Disappearance of clones over the observed time period was rare, with just 3 of 44 clones falling below detection in follow-up samples.

Figure 2. Effect of Mutation Order on Disease Phenotype

In Panel A, the pie chart shows the order of acquisition of mutations in 24 patients. Half the patients with myeloproliferative neoplasms acquired the JAK2 mutation first (JAK2-first) and half acquired the TET2 mutation first (TET2-first). Both orders of acquisition of mutations occur in each disease subtype. ET denotes essential thrombocythemia, MF myelofibrosis, and PV polycythemia vera. In Panel B, the bar graph shows the proportion of JAK2 V617F–homozygous colonies in the total number of mutant colonies. Patients in whom JAK2 V617F was acquired first had a greater proportion of homozygous colonies in the mutant compartment (mean percentage of homozygous mutant colonies in JAK2-first patients, 57.8%, vs. 1.4% in TET2-first patients; P<0.001 by the t-test). Patients are indicated by their patient-identification numbers. T bars indicate standard errors. In Panel C, the bar graph shows the relative number of progenitor cells in the TET2-first patients and JAK2-first patients. The three bars represent the relative number of common myeloid progenitors (CMPs) (lin⁻CD34⁺CD38⁺CD90⁻ CD10⁻FLK2⁺CD45RA⁻), granulocyte–macrophage progenitors (GMPs) (lin⁻CD34⁺CD38⁺CD90⁻CD10⁻FLK2⁺CD45RA⁺), and megakaryocyte–erythroid progenitors (MEPs) (lin⁻CD34⁺CD38⁺CD90⁻CD10⁻FLK2⁻CD45RA⁻). TET2-first patients had a uniform predominance of CMPs over other progenitors within the CD34⁺CD38⁺ compartment (P = 0.001 by the t-test). By contrast, in JAK2-first patients, MEPs were more prevalent than CMPs or GMPs (P<0.001 by the t-test). When the samples were obtained, the ages of the TET2-first and JAK2-first patients were similar (mean age, 78 years [range, 66 to 86] vs. 72 years [range, 50 to 84]).

Figure 3. Influence of Mutation Order on Proliferation of Stem and Progenitor Cells and Expression of Progenitor Genes

In Panel A, the bar graph shows the average number of cells (clone size) that were present at 10 days in cultures of hematopoietic stem and progenitor cells (HSPCs) of different genotypes. Bars represent nonmutant, single-mutant, and double-mutant (both TET2 and JAK2) clones. In the three TET2-first patients, the size of TET2 single-mutant clones did not differ significantly from that of nonmutant clones (P = 0.24 by the t-test) or double-mutant clones (P = 0.07 by the t-test). By contrast, in the four JAK2-first patients, JAK2 single-mutant clones were significantly larger than nonmutant clones (P = 0.05 by the t-test) and double-mutant clones (P = 0.04 by the t-test). T bars indicate standard errors, single asterisks P<0.05, and double asterisks P<0.01. In Panel B, the number of secondary colony-forming cells created per HSPC is shown for two patients who had a TET2 mutation followed by a JAK2 V617F mutation and four patients who had a JAK2 mutation followed by a TET2 mutation. HSPCs that acquire JAK2 V617F on a TET2-mutant background, as compared with TET2 single-mutant HSPCs, produce fewer colony-forming cells (mean, 3 colony-forming cells vs. 12 colony-forming cells; P = 0.002 by the t-test), whereas those that acquire TET2 mutations on a JAK2 single-mutant background, as compared with JAK2 single-mutant HSPCs, produce more colony-forming cells (mean, 27 colony-forming cells vs. 16 colony-forming cells; P = 0.01 by the t-test). In Panel C, each bar graph shows the percentage of total HSPCs of each genotype measured in five TET2-first patients and six JAK2-first patients. The percentage of HSPCs that retained the first mutation alone was higher in TET2-first patients than in JAK2-first patients. In Panel D, each bar graph shows the percentage of BFU-E colonies of each genotype in five TET2-first patients and six JAK2-first patients. In contrast to the proportion of mutant HSPCs (Panel C), the TET2-first BFU-E compartment is skewed toward the double-mutant clone. The JAK2-first BFU-E compartment is similar to the HSPC compartment. In Panel E, each Venn diagram shows the overlap in genes that are commonly up-regulated (left diagram, 54 common genes) or down-regulated (right diagram, 61 common genes) when JAK2 V617F is acquired on different backgrounds. In Panel F, the Venn diagram shows the numbers of genes that are up-regulated when JAK2 V617F is acquired on a nonmutant background overlapped with those that are down-regulated when JAK2 V617F is acquired on a TET2-mutant background. The 10 genes that followed this pattern are listed in the box.

Figure 4. Clinical Significance of Mutation Order in Myeloproliferative Neoplasms

In Panel A, the mean age at presentation of 48 patients with myeloproliferative neoplasms in whom mutation order was determined is shown. On average, TET2-first patients presented 10.46 years later than JAK2-first patients (P = 0.002 by the t-test; P = 0.02 by two-way analysis of variance accounting for order of acquisition of mutation, sex, white-cell count, and phenotype of the myeloproliferative neoplasm). I bars indicate standard errors, and double asterisks P<0.01. In Panel B, histograms represent the number of patients who received a diagnosis of essential thrombocythemia (ET) or polycythemia vera (PV). JAK2-first patients were more likely to receive a diagnosis of PV (P = 0.05 by the chi-square test). The single asterisk indicates P<0.05. In Panel C, a Kaplan–Meier curve shows thrombosis-free survival among all 48 patients in whom mutation order was determined. JAK2-first status was identified as an independent risk factor for thrombotic events in addition to the known risk factor of age (P = 0.002 by multivariate analysis for mutational order and P = 0.01 by Cox proportional-hazards model for age taking into account age, prior thrombosis, sex, subtype of myeloproliferative neoplasm, white-cell count, receipt of cytoreductive therapy, and order of mutations). In Panel D, the graph shows the relative sensitivity of colonies with distinct genotypes to ruxolitinib in a colony-forming cell assay (4 TET2-first patients and 4 JAK2-first patients, 35 to 66 colonies picked per patient). In TET2-first patients, single-mutant colonies had a TET2 mutation alone, and in JAK2-first patients, single-mutant colonies had a JAK2-heterozygous or JAK2-homozygous mutation. Single-mutant clones in JAK2-first patients were more sensitive to ruxolitinib (left side, P<0.01 by the one-tailed t-test). Double-mutant clones (those bearing both JAK2 and TET2 mutations) in JAK2-first patients were also more sensitive to ruxolitinib as compared with clones from TET2-first patients (right side, P = 0.03 by the one-tailed t-test). In Panel E, the way in which the order of mutation acquisition influences the evolution of disease is shown. This model depicts the manner in which single hematopoietic units (left), consisting of stem cells, progenitors, and differentiated cells, acquire mutations over time. Some units are hyperproliferative and produce excess differentiated cells that contribute to the disease phenotype. The numbers represent the acquisition of the first mutation (1), second mutation (2), and JAK2 V617F homozygosity (3). Patients who acquire a TET2 mutation first gain a self-renewal advantage but do not over-produce downstream progeny. The expansion of the TET2-alone clone (bold borders) without excess differentiated cells leads to clonal expansion without immediate clinical presentation. Hematopoietic stem cells that acquire a secondary JAK2 mutation (pink fill) compete with the TET2-alone clone, and their increased proliferation at the progenitor level drives an overproduction of terminal cells. When homozygosity is acquired as a third event (red fill), this clone has limited space to expand because of the high self-renewal activity of TET2-alone and TET2–JAK2–heterozygous clones. Patients who acquire a JAK2 mutation first (pink fill, lower panel) produce excess differentiated cells in the absence of a distinct self-renewal advantage in the hematopoietic stem cells. When a secondary TET2 mutation is acquired, hematopoietic stem cells obtain a self-renewal advantage and JAK2–TET2–mutant cells expand at the stem-cell level. Hematopoietic stem cells with loss of heterozygosity of JAK2 V617F (acquired before or after the TET2 mutation) (red fill) also have space to expand and result in a more pronounced excess of differentiated cells. This excess production would explain both the presentation as a polycythemia vera and the elevated risk of thrombotic events in JAK2-first patients.