Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.)

Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.)

Selective sequencing or in silico analysis for differences in DNA fragment size can improve the detection of circulating tumor DNA.

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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. 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. 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. 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.)

Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication.