Abstract Mutations in cancer-associated genes drive tumour outgrowth. However, the timing of driver mutations and dynamics of clonal expansion that lead to human cancers are largely unknown. We used 448,553 somatic mutations from whole-genome sequencing of 843 clonal haematopoietic colonies to reconstruct the phylogeny of haematopoiesis, from embryogenesis to clinical disease, in 10 patients with myeloproliferative neoplasms which are blood cancers more common in older age. JAK2V617F, the pathognomonic mutation in these cancers, was acquired in utero or childhood, with upper estimates of age of acquisition ranging between 4.1 months and 11.4 years across 5 patients. DNMT3A mutations, which are associated with age-related clonal haematopoiesis, were also acquired in utero or childhood, by 7.9 weeks of gestation to 7.8 years across 4 patients. Subsequent driver mutation acquisition was separated by decades. The mean latency between JAK2V617F acquisition and clinical presentation was 34 years (range 20-54 years). Rates of clonal expansion varied substantially (<10% to >200% expansion/year), were affected by additional driver mutations, and predicted latency to clinical presentation. Driver mutations and rates of expansion would have been detectable in blood one to four decades before clinical presentation. This study reveals how driver mutation acquisition very early in life with life-long growth trajectories drive adult blood cancer, providing opportunities for early detection and intervention, and a new paradigm for cancer development.
ABSTRACT Mutations in cancer-associated genes drive tumour outgrowth. However, the timing of driver mutations and dynamics of clonal expansion that lead to human cancers are largely unknown. We used 448,553 somatic mutations from whole-genome sequencing of 843 clonal haematopoietic colonies to reconstruct the phylogeny of haematopoiesis, from embryogenesis to clinical disease, in 10 patients with myeloproliferative neoplasms which are blood cancers more common in older age. JAK2 V617F , the pathognomonic mutation in these cancers, was acquired in utero or childhood, with upper estimates of age of acquisition ranging between 4.1 months and 11.4 years across 5 patients. DNMT3A mutations, which are associated with age-related clonal haematopoiesis, were also acquired in utero or childhood, by 7.9 weeks of gestation to 7.8 years across 4 patients. Subsequent driver mutation acquisition was separated by decades. The mean latency between JAK2 V617F acquisition and clinical presentation was 31 years (range 12-54 years). Rates of clonal expansion varied substantially (<10% to >200% expansion/year), were affected by additional driver mutations, and predicted latency to clinical presentation. Driver mutations and rates of expansion would have been detectable in blood one to four decades before clinical presentation. This study reveals how driver mutation acquisition very early in life with life-long growth and evolution drive adult blood cancer, providing opportunities for early detection and intervention, and a new paradigm for cancer development.
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.).
Abstract Current therapies for myeloproliferative neoplasms (MPN) improve symptoms but have limited effect on tumor size. In preclinical studies, tamoxifen restored normal apoptosis in mutated hematopoietic stem and progenitor cells (HSPCs). TAMARIN is a Phase-II, multicenter, single-arm clinical trial assessing tamoxifen’s safety and activity in patients with stable MPNs, no prior thrombotic events and mutated JAK2V617F, CALRins5 or CALRdel52 peripheral blood allele burden ≥20%. The primary outcome (≥50% allele burden reduction at 24 weeks) was met by 3/38 patients; 5/38 additional patients showed ≥25% reductions. Tamoxifen was well tolerated. Baseline analysis of HSPC transcriptome segregated responders and non-responders, suggesting a predictive signature. In responder HSPCs, longitudinal analysis showed high baseline expression of JAK-STAT signaling and oxidative phosphorylation genes, which were downregulated by tamoxifen. In JAK2V617F+ cells, 4-hydroxytamoxifen inhibited mitochondrial complex-I, activating proapoptotic integrated stress response (ISR) and decreasing pathogenic JAK2 signaling. Therefore, tamoxifen inhibits mitochondrial respiration, modulates ISR and suppresses pathogenic JAK-STAT signaling in a subset of prospectively identifiable MPN patients.
In a recent issue of HemaSphere, Luque Paz and colleagues1 described the reconstruction of clonal architecture in myeloproliferative neoplasms (MPN) using single-cell DNA sequencing (scDNAseq). This approach not only had similar power to traditional methodologies based on the growth and sequencing of individual hematopoietic colonies, but distinct patterns of clonal hierarchy were also reported to show prognostic significance. The ease of studying single-cell-derived hematopoietic colonies, most often grown from peripheral blood, has been a long-standing foundation and strength of MPN biological research. Early colony studies of patients with JAK2 mutations, for example, identified the differing patterns of JAK2 V617F heterozygosity and homozygosity between patients with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF)2 and confirmed the presence of mutations in almost all patients in PV.3 Following the identification of other gene mutations in myeloid disease, analysis of comutation patterns within colonies then illuminated the clonal dynamics of stable and progressive MPN: that complex, branching or oligoclonal substructures could exist even in chronic phase4–7; that the same 2 mutations acquired linearly but in different orders could have variable biological consequences8; and that specific subclonal genomic events could drive disease transformation.7,9 Although a backbone of many important MPN studies, sequencing of many thousands of individual hematopoietic colonies is labor-intensive and could never be applicable to routine clinical practice. Luque Paz and colleagues used the alternative method of scDNAseq (Tapestri technology), used previously in other myeloid malignancies particularly acute myeloid leukemia (AML),10 to sequence the individual subclones present in MPN peripheral blood samples.1 A total of 50 MPN patients with JAK2 V617F and additional mutations were studied with traditional liquid or methylcellulose colony assays and the clonal substructures delineated were compared with results from scDNAseq in 22 of these. Although almost all mutations originally identified in bulk DNA were detected by both methods, there was 80% concordance in detection of individual clones and subclones. Although scDNAseq seems less reliable in distinguishing between heterozygous and homozygous mutant subclones, a broad scDNAseq panel can detect additional low-level clones whose variants might not be detected when sequencing bulk granulocyte DNA. scDNAseq also avoids the risk of differential clonal efficiencies in culture leading to overrepresentation or underrepresentation of specific genotypes. scDNAseq can, therefore, comprehensively decode the subclonal architecture of a broad panel of variants from a single peripheral blood sample without the labor-intensive processes involved in culturing and genotyping numerous colonies. Based on the clonal architecture data from all 50 patients, 4 patterns (clusters) were identified in an unsupervised clustering analysis, differing in number of mutations, architectural complexity including comutation within subclones, order of mutation acquisition, and presence of JAK2 V617F homozygosity. The group with the most complex clonal architecture (cluster 4) showed the highest rate of transformation to AML/MDS and a reduced overall survival, which was independent of MPN subtype, age at diagnosis, and the presence of high molecular risk mutations. By contrast, a group (cluster 1) who carried additional mutations but mostly in a different clone to the JAK2 V617F—although these included high-risk mutations such as in ASXL1 or EZH2—had the best overall survival. Genomic data have become an integral part of prognostic modeling in all MPN subtypes11–13 (Figure 1). Previous studies have demonstrated that a higher total number of mutations is associated with increased transformation and poorer survival7; a higher number of high molecular risk mutations is also associated with reduced survival in myelofibrosis.11 This new study now delivers the clear message that the prognosis of a MPN does not simply reflect the sum of its genomic abnormalities, but the way in which these interact within a clonal substructure. In particular for cluster 1, in whom most additional mutations were not found within the JAK2-mutant clone, the predictive power of the total number of mutations was actually improved when these mutations outside the JAK2-mutant clone were discarded altogether. Moreover, the clonal architecture-based classification outperformed the Sanger risk calculator13 in predicting outcome for this group.Figure 1.: Evolution of prognostic modeling in the myeloproliferative neoplasms. 11–21It is well recognized that mutations can cooperate biologically in myeloid disease, making it logical that patterns of comutation are highly relevant clinically. Certain genes show increased likelihood of comutation in AML, myelodysplastic neoplasms, or MPN.13,22,23 With JAK2 V617F, combination with TET2 loss, EZH2 loss or gain of mutant IDH1/2 have all been shown to have synergistic effects, driving more severe myeloproliferative phenotypes in mouse models through effects on hematopoietic stem and progenitor cell function and differentiation.24–26 Moreover, the phenomenon of clonal hematopoiesis of indeterminate potential, where small hemopoietin subclones carrying isolated mutations in genes such as ASXL1 can exist in the absence of any definite hematological neoplasm,27 is consistent with the finding that such subclones, if entirely separate to the JAK2-mutant clone, might not adversely affect the outcome of a patient with MPN at all. How might these findings be translated to clinical practice? The prognostic model requires independent validation and it is important to note that only a subgroup of MPN patients were covered, specifically those with JAK2 V617F and additional mutation(s). The utility of such a model in patients with CALR- or MPL-mutant disease requires further exploration and no patients with a single driver were included. The incremental benefit of the clonal architecture model was also mainly seen in a group of patients (cluster 1) that included many with ET and PV, in whom comprehensive prognostic profiling is often not clinically necessary at present. Besides these caveats, cost would currently prevent the widespread use of scDNAseq, although in recent years other technologies previously considered prohibitively expensive have transitioned to routine diagnostic processes.28 Cancer diagnostics is increasingly moving toward establishing each individual patient’s full genomic profile and using this as a surrogate for disease biology to inform diagnosis, personalized prognostic predictions, and, where possible, personalized therapy. In other fields, broad techniques including whole genome sequencing have advanced clinical practice through the ability to map a cancer’s genomic lesions comprehensively.28 Chronic myeloid malignancies have some particular considerations: clinical practice includes decisions about whether to treat at all and therapeutic studies are already considering whether treatments might drive regression, outgrowth, or evolution of disease subclones.29 In this context, the ability to generate an unbiased snapshot of clonal architecture from peripheral blood on diagnosis, follow-up, and progression is appealing. If affordable and deliverable clinically in future, scDNAseq technologies could therefore not only have a role in further evolution from current prognostic models (Figure 1), but also facilitate studies of how best to tailor therapy for individual MPN patients. AUTHOR CONTRIBUTIONS ALG conceptualized and wrote the article. DISCLOSURES The author has no conflicts of interest to disclose. SOURCES OF FUNDING The author has no sources of funding to disclose for this manuscript.
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