Life histories of myeloproliferative neoplasms inferred from phylogenies.

Mutations in cancer-associated genes drive tumour outgrowth, but our knowledge of the timing of driver mutations and subsequent clonal dynamics is limited1-3. Here, using whole-genome sequencing of 1,013 clonal haematopoietic colonies from 12 patients with myeloproliferative neoplasms, we identified 580,133 somatic mutations to reconstruct haematopoietic phylogenies and determine clonal histories. Driver mutations were estimated to occur early in life, including the in utero period. JAK2V617F was estimated to have been acquired by 33 weeks of gestation to 10.8 years of age in 5 patients in whom JAK2V617F was the first event. DNMT3A mutations were acquired by 8 weeks of gestation to 7.6 years of age in 4 patients, and a PPM1D mutation was acquired by 5.8 years of age. Additional genomic events occurred before or following JAK2V617F acquisition and as independent clonal expansions. Sequential driver mutation acquisition was separated by decades across life, often outcompeting ancestral clones. The mean latency between JAK2V617F acquisition and diagnosis was 30 years (range 11-54 years). Estimated historical rates of clonal expansion varied substantially (3% to 190% per year), increased with additional driver mutations, and predicted latency to diagnosis. Our study suggests that early driver mutation acquisition and life-long growth and evolution underlie adult myeloproliferative neoplasms, raising opportunities for earlier intervention and a new model for cancer development.

Outcomes and characteristics of nonmelanoma skin cancers in patients with myeloproliferative neoplasms on ruxolitinib.

ABSTRACT: Nonmelanoma skin cancers (NMSCs) in ruxolitinib-treated patients with myeloproliferative neoplasms behave aggressively, with adverse features and high recurrence. In our cohort, mortality from metastatic NMSC exceeded that from myelofibrosis. Vigilant skin assessment, counseling on NMSC risks, and prospective ruxolitinib-NMSC studies are crucial.

Essential thrombocythemia: challenges in clinical practice and future prospects.

Essential thrombocythemia (ET) was first described in 1934, and subsequently, progress has been made in better understanding the molecular pathogenesis and which patients may have greatest risk of progression or vascular events. However, it has been more than a decade since a new therapy has been approved for ET. We are beginning to understand more comprehensively both the heterogeneity of this disease, which is largely driven by driver mutation status, as well as the effect of disease-related symptoms, such as fatigue, on patients. In this review we provide a practical overview of diagnosis and management of ET with focus on challenging patient scenarios and some consideration of what comprehensive care might entail. Finally, we also discuss newer therapies and how these might be assessed.

Classification and Personalized Prognosis in Myeloproliferative Neoplasms.

BACKGROUND: Myeloproliferative neoplasms, such as polycythemia vera, essential thrombocythemia, and myelofibrosis, are chronic hematologic cancers with varied progression rates. The genomic characterization of patients with myeloproliferative neoplasms offers the potential for personalized diagnosis, risk stratification, and treatment. METHODS: We sequenced coding exons from 69 myeloid cancer genes in patients with myeloproliferative neoplasms, comprehensively annotating driver mutations and copy-number changes. We developed a genomic classification for myeloproliferative neoplasms and multistage prognostic models for predicting outcomes in individual patients. Classification and prognostic models were validated in an external cohort. RESULTS: A total of 2035 patients were included in the analysis. A total of 33 genes had driver mutations in at least 5 patients, with mutations in JAK2, CALR, or MPL being the sole abnormality in 45% of the patients. The numbers of driver mutations increased with age and advanced disease. Driver mutations, germline polymorphisms, and demographic variables independently predicted whether patients received a diagnosis of essential thrombocythemia as compared with polycythemia vera or a diagnosis of chronic-phase disease as compared with myelofibrosis. We defined eight genomic subgroups that showed distinct clinical phenotypes, including blood counts, risk of leukemic transformation, and event-free survival. Integrating 63 clinical and genomic variables, we created prognostic models capable of generating personally tailored predictions of clinical outcomes in patients with chronic-phase myeloproliferative neoplasms and myelofibrosis. The predicted and observed outcomes correlated well in internal cross-validation of a training cohort and in an independent external cohort. Even within individual categories of existing prognostic schemas, our models substantially improved predictive accuracy. CONCLUSIONS: Comprehensive genomic characterization identified distinct genetic subgroups and provided a classification of myeloproliferative neoplasms on the basis of causal biologic mechanisms. Integration of genomic data with clinical variables enabled the personalized predictions of patients' outcomes and may support the treatment of patients with myeloproliferative neoplasms. (Funded by the Wellcome Trust and others.).

Effect of mutation order on myeloproliferative neoplasms.

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.).

JAK2V617F promotes replication fork stalling with disease-restricted impairment of the intra-S checkpoint response.

Cancers result from the accumulation of genetic lesions, but the cellular consequences of driver mutations remain unclear, especially during the earliest stages of malignancy. The V617F mutation in the JAK2 non-receptor tyrosine kinase (JAK2V617F) is present as an early somatic event in most patients with myeloproliferative neoplasms (MPNs), and the study of these chronic myeloid malignancies provides an experimentally tractable approach to understanding early tumorigenesis. Introduction of exogenous JAK2V617F impairs replication fork progression and is associated with activation of the intra-S checkpoint, with both effects mediated by phosphatidylinositide 3-kinase (PI3K) signaling. Analysis of clonally derived JAK2V617F-positive erythroblasts from MPN patients also demonstrated impaired replication fork progression accompanied by increased levels of replication protein A (RPA)-containing foci. However, the associated intra-S checkpoint response was impaired in erythroblasts from polycythemia vera (PV) patients, but not in those from essential thrombocythemia (ET) patients. Moreover, inhibition of p53 in PV erythroblasts resulted in more gamma-H2Ax (γ-H2Ax)-marked double-stranded breaks compared with in like-treated ET erythroblasts, suggesting the defective intra-S checkpoint function seen in PV increases DNA damage in the context of attenuated p53 signaling. These results demonstrate oncogene-induced impairment of replication fork progression in primary cells from MPN patients, reveal unexpected disease-restricted differences in activation of the intra-S checkpoint, and have potential implications for the clonal evolution of malignancies.

JAK2V617F homozygosity drives a phenotypic switch in myeloproliferative neoplasms, but is insufficient to sustain disease.

Genomic regions of acquired uniparental disomy (UPD) are common in malignancy and frequently harbor mutated oncogenes. Homozygosity for such gain-of-function mutations is thought to modulate tumor phenotype, but direct evidence has been elusive. Polycythemia vera (PV) and essential thrombocythemia (ET), 2 subtypes of myeloproliferative neoplasms, are associated with an identical acquired JAK2V617F mutation but the mechanisms responsible for distinct clinical phenotypes remain unclear. We provide direct genetic evidence and demonstrate that homozygosity for human JAK2V617F in knock-in mice results in a striking phenotypic switch from an ET-like to PV-like phenotype. The resultant erythrocytosis is driven by increased numbers of early erythroid progenitors and enhanced erythroblast proliferation, whereas reduced platelet numbers are associated with impaired platelet survival. JAK2V617F-homozygous mice developed a severe hematopoietic stem cell defect, suggesting that additional lesions are needed to sustain clonal expansion. Together, our results indicate that UPD for 9p plays a causal role in the PV phenotype in patients as a consequence of JAK2V617F homozygosity. The generation of a JAK2V617F allelic series of mice with a dose-dependent effect on hematopoiesis provides a powerful model for studying the consequences of mutant JAK2 homozygosity.

JAK2V617F homozygosity arises commonly and recurrently in PV and ET, but PV is characterized by expansion of a dominant homozygous subclone.

Subclones homozygous for JAK2V617F are more common in polycythemia vera (PV) than essential thrombocythemia (ET), but their prevalence and significance remain unclear. The JAK2 mutation status of 6495 BFU-E, grown in low erythropoietin conditions, was determined in 77 patients with PV or ET. Homozygous-mutant colonies were common in patients with JAK2V617F-positive PV and were surprisingly prevalent in JAK2V617F-positive ET and JAK2 exon 12-mutated PV. Using microsatellite PCR to map loss-of-heterozygosity breakpoints within individual colonies, we demonstrate that recurrent acquisition of JAK2V617F homozygosity occurs frequently in both PV and ET. PV was distinguished from ET by expansion of a dominant homozygous subclone, the selective advantage of which is likely to reflect additional genetic or epigenetic lesions. Our results suggest a model in which development of a dominant JAK2V617F-homzygous subclone drives erythrocytosis in many PV patients, with alternative mechanisms operating in those with small or undetectable homozygous-mutant clones.

Inherited polygenic effects on common hematological traits influence clonal selection on JAK2V617F and the development of myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are chronic cancers characterized by overproduction of mature blood cells. Their causative somatic mutations, for example, JAK2V617F, are common in the population, yet only a minority of carriers develop MPN. Here we show that the inherited polygenic loci that underlie common hematological traits influence JAK2V617F clonal expansion. We identify polygenic risk scores (PGSs) for monocyte count and plateletcrit as new risk factors for JAK2V617F positivity. PGSs for several hematological traits influenced the risk of different MPN subtypes, with low PGSs for two platelet traits also showing protective effects in JAK2V617F carriers, making them two to three times less likely to have essential thrombocythemia than carriers with high PGSs. We observed that extreme hematological PGSs may contribute to an MPN diagnosis in the absence of somatic driver mutations. Our study showcases how polygenic backgrounds underlying common hematological traits influence both clonal selection on somatic mutations and the subsequent phenotype of cancer.

Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations.

Molecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 1-3%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results.

Clinical significance of SF3B1 mutations in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.

In a previous study, we identified somatic mutations of SF3B1, a gene encoding a core component of RNA splicing machinery, in patients with myelodysplastic syndrome (MDS). Here, we define the clinical significance of these mutations in MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). The coding exons of SF3B1 were screened using massively parallel pyrosequencing in patients with MDS, MDS/MPN, or acute myeloid leukemia (AML) evolving from MDS. Somatic mutations of SF3B1 were found in 150 of 533 (28.1%) patients with MDS, 16 of 83 (19.3%) with MDS/MPN, and 2 of 38 (5.3%) with AML. There was a significant association of SF3B1 mutations with the presence of ring sideroblasts (P < .001) and of mutant allele burden with their proportion (P = .002). The mutant gene had a positive predictive value for ring sideroblasts of 97.7% (95% confidence interval, 93.5%-99.5%). In multivariate analysis including established risk factors, SF3B1 mutations were found to be independently associated with better overall survival (hazard ratio = 0.15, P = .025) and lower risk of evolution into AML (hazard ratio = 0.33, P = .049). The close association between SF3B1 mutations and disease phenotype with ring sideroblasts across MDS and MDS/MPN is consistent with a causal relationship. Furthermore, SF3B1 mutations are independent predictors of favorable clinical outcome, and their incorporation into stratification systems might improve risk assessment in MDS.

Clonal analyses reveal associations of JAK2V617F homozygosity with hematologic features, age and gender in polycythemia vera and essential thrombocythemia.

Subclones homozygous for JAK2V617F are more common and larger in patients with polycythemia vera compared to essential thrombocythemia, but their role in determining phenotype remains unclear. We genotyped 4564 erythroid colonies from 59 patients with polycythemia vera or essential thrombocythemia to investigate whether the proportion of JAK2V617F -homozygous precursors, compared to heterozygous precursors, is associated with clinical or demographic features. In polycythemia vera, a higher proportion of homozygous-mutant precursors was associated with more extreme blood counts at diagnosis, consistent with a causal role for homozygosity in polycythemia vera pathogenesis. Larger numbers of homozygous-mutant colonies were associated with older age, and with male gender in polycythemia vera but female gender in essential thrombocythemia. These results suggest that age promotes development or expansion of homozygous-mutant clones and that gender modulates the phenotypic consequences of JAK2V617F homozygosity, thus providing a potential explanation for the long-standing observations of a preponderance of men with polycythemia vera but of women with essential thrombocythemia.

Clonal origin of KMT2A wild-type lineage-switch leukemia following CAR-T cell and blinatumomab therapy.

Children with acute lymphoblastic leukemia (ALL) undergoing anti-CD19 therapy occasionally develop acute myeloid leukemia (AML). The clonal origin of such lineage-switch leukemias1-4 remains unresolved. Here, we reconstructed the phylogeny of multiple leukemias in a girl who, following multiply relapsed ALL, received anti-CD19 cellular and antibody treatment and subsequently developed AML. Whole genome sequencing unambiguously revealed the AML derived from the initial ALL, with distinct driver mutations that were detectable before emergence. Extensive prior diversification and subsequent clonal selection underpins this fatal lineage switch. Genomic monitoring of primary leukemias and recurrences may predict therapy resistance, especially regarding anti-CD19 treatment.

Continuous Indexing of Fibrosis (CIF): improving the assessment and classification of MPN patients.

The grading of fibrosis in myeloproliferative neoplasms (MPN) is an important component of disease classification, prognostication and monitoring. However, current fibrosis grading systems are only semi-quantitative and fail to fully capture sample heterogeneity. To improve the quantitation of reticulin fibrosis, we developed a machine learning approach using bone marrow trephine (BMT) samples (n = 107) from patients diagnosed with MPN or a reactive marrow. The resulting Continuous Indexing of Fibrosis (CIF) enhances the detection and monitoring of fibrosis within BMTs, and aids MPN subtyping. When combined with megakaryocyte feature analysis, CIF discriminates between the frequently challenging differential diagnosis of essential thrombocythemia (ET) and pre-fibrotic myelofibrosis with high predictive accuracy [area under the curve = 0.94]. CIF also shows promise in the identification of MPN patients at risk of disease progression; analysis of samples from 35 patients diagnosed with ET and enrolled in the Primary Thrombocythemia-1 trial identified features predictive of post-ET myelofibrosis (area under the curve = 0.77). In addition to these clinical applications, automated analysis of fibrosis has clear potential to further refine disease classification boundaries and inform future studies of the micro-environmental factors driving disease initiation and progression in MPN and other stem cell disorders.