Order Matters: The Order of Somatic Mutations Influences Cancer Evolution

Abstract

Cancers evolve as a consequence of multiple somatic lesions, with competition between subclones and sequential subclonal evolution. Some driver mutations arise either early or late in the evolution of different individual tumors, suggesting that the final malignant properties of a subclone reflect the sum of mutations acquired rather than the order in which they arose. However, very little is known about the cellular consequences of altering the order in which mutations are acquired. Recent studies of human myeloproliferative neoplasms show that the order in which individual mutations are acquired has a dramatic impact on the cell biological and molecular properties of tumor-initiating cells. Differences in clinical presentation, complications, and response to targeted therapy were all observed and implicate mutation order as an important player in cancer biology. These observations represent the first demonstration that the order of mutation acquisition influences stem and progenitor cell behavior and clonal evolution in any cancer. Thus far, the impact of different mutation orders has only been studied in hematological malignancies, and analogous studies of solid cancers are now required.

Figure 1.

Determining the mutation order in hematological malignancies. (A) Different scenarios for how two mutations could lead to cancer with the only difference being the order in which mutations are acquired. (B) How the colony assay can be used to determine mutation order. Briefly, single colonies are grown from peripheral blood or bone marrow samples and individually picked and sequenced for JAK2 and TET2 mutations. The presence of a single-mutant clone for one of these genes allows determination of mutation order as displayed.

Figure 2.

Order of mutation acquisition influences the evolution of disease. 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 overproduce 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. (Both the figure and legend are from Ortmann et al. 2015, Massachusetts Medical Society; reprinted, with permission, © 2015.)

Figure 3.

Potential mechanisms for how mutation order alters disease evolution. (A) The intrinsic epigenetic impact of mutation order. Here, the first mutation impacts the accessibility of particular genomic loci such that the second mutation cannot make the changes it would normally make on a wild-type (WT) background. In this example, black circles represent chromatin compaction by histones induced by mutation B that block the accessibility of a promoter region for a particular transcription factor (green oval) that is stimulated by mutation A. The gene would be turned ON if mutation A came first and OFF if B came first. (B) How a single mutation could create a different collection of target cells. In this case, mutation A induces rapid cell growth and differentiation from the initial cell, creating many distinct target cell types, whereas mutation B creates a slower growing, more undifferentiated clone. The second mutation could therefore occur in a different target cell depending on which mutation comes first. (C) The extrinsic environmental impact of mutation order. Here the first mutation (A or B) gives rise to different numbers and types of mature cells, thereby altering the cellular environment that the double-mutant clone (AB) finds itself when it is first created. These distinct cellular environments may contribute to distinct disease evolution.