A) Heterogeneity can arise within tumors through stochastic genetic () and epigenetic () changes that confer heritable phenotypic and functional differences upon cancer cells. This process is known as clonal evolution because the genetic/epigenetic changes are subject to selection within tumors. This process tends to lead to more aggressive cancers over time; however, some cancer cells (grey) would be predicted to lose their tumorigenic capacity as a consequence of disadvantageous genetic changes. B) Heterogeneity can arise in response to extrinsic environmental differences within tumors: cancer cells (blue) adjacent to blood vessels (red) are different from cancer cells further from blood vessels (white) (). The differences are shown as being reversible, though environmental differences could also cause irreversible changes in cancer cell properties. C) Cancers that follow the stem cell model contain intrinsically different subpopulations of tumorigenic (red) and non-tumorigenic cells (yellow and green) organized in a hierarchy in which a minority population of tumorigenic cells gives rise to phenotypically diverse non-tumorigenic cells. Non-tumorigenic cells are thought to compose the bulk of tumors but have little capacity to contribute to cancer progression (; ; ). Tumorigenic cells can be serially transplanted, re-establishing phenotypic heterogeneity with each passage. D) Cancers that follow the stem cell model are also subject to clonal evolution as well as heterogeneity from environmental differences within tumors. Thus, these sources of heterogeneity are not mutually exclusive and may each apply to variable extents depending on the cancer.

Differences in the cell-of-origin can directly and indirectly influence the phenotype of tumorigenic cells and, perhaps, whether or not the cancer is hierarchically organized. A) Different cell types in a stem/progenitor cell hierarchy within a normal tissue may be transformed into cancer cells. The properties of the cell-of-origin influence the types of mutations that are competent to transform and the properties of the resulting cancer (; ). (B) Spatial differences in the identity of the cell-of-origin within tissues influence the types of mutations that are competent to transform and the properties of the resulting cancer (; ). (C) Temporal differences in the cell-of-origin also influence the types of mutations that are competent to transform and the properties of the resulting cancer (Magee and Morrison, unpublished data), consistent with the observation that the driver mutation spectrum changes with age in patients ()(see text for references regarding age-related changes in the incidence of specific mutations).

Although most AMLs follow a cancer stem cell model, the surface marker phenotypes of the leukemogenic cells vary from patient to patient. (A–C) Different oncogenic mutations can transform cells at different levels within the hematopoiesis hierarchy (), potentially influencing the frequency, spectrum, and phenotype of cells with leukemogenic potential. Interconversion between leukemogenic cell populations would allow any population to recapitulate the heterogeneity of the leukemogenic cell pool (; ). (D) Some mutations, such as MLL-AF9 translocations, can confer leukemogenic activity upon restricted progenitors (; ; ; ). (E) If multiple populations have leukemogenic capacity but do not interconvert, then only the most immature cells can recapitulate the full heterogeneity of the parent leukemia but multiple levels of the hierarchy may be able to drive disease progression (). (F) In some ALLs many cells have leukemogenic activity despite heterogeneity in marker expression (; ).

A) Potential describes what cells can do in a permissive environment. The cancer stem cell model, and the transplantation assays (black arrows) on which it is largely based, address the potential of cancer cells to form tumors. B) Fate reflects what cells actually do in a specific environment. In the context of cancer, the question is which cells are fated to contribute to tumor growth and disease progression in their actual environment in the patient. Many of the cells that have the potential to form tumors upon transplantation may not be fated to contribute to disease progression in a particular patient because they are not in a permissive environment. For example, some cancer cells undergo cell death due to hypoxia or immune effector activity. Some of these cells might have the potential to form a tumor if transplanted into another environment, but are fated to undergo cell death in the tumor environment in which they actually reside in the patient. There may also be cells that lack the ability to form a tumor upon transplantation but that are nonetheless fated to contribute to disease progression in the patient, such as if they acquire a new mutation that increases their proliferation. Transplantation assays only assess potential, not fate in a patient, and therefore should attempt to detect the full range of cells with the potential to form tumors. No transplantation assay mimics the environment within patient tissues and such assays are neither designed nor capable of assessing cell fate in patients. Consequently, very little is known about the spectrum of cells fated to contribute to tumor growth and disease progression in patients, or the extent to which it overlaps with the spectrum of cells that can form a tumor upon transplantation.

A) According to the clonal evolution model many cancer cells have tumorigenic potential (circular self-renewal arrows) and heterogeneity arises through stochastic genetic/epigenetic changes (lightning bolt). Changes in cell phenotype are not necessarily associated with changes in tumorigenic potential. B) For cancers that follow a stem cell model in which only the cells at the top of the hierarchy retain tumorigenic capacity, the differentiation of these cells into non-tumorigenic progeny creates tumor heterogeneity. Differentiation is associated with a loss of tumorigenic potential. C) For cancers with a high rate of genetic change, clones of cells within the hierarchy depicted in panel B may acquire tumorigenic potential as a consequence of new mutations. Phenotypic changes are sometimes associated with changes in tumorigenic potential and sometimes not. Note that it would be difficult to experimentally distinguish this model from the model in panel A. D) A cancer that is hierarchically organized according to the cancer stem cell model but in which non-tumorigenic cells can inefficiently revert to higher levels of the hierarchy. In this case, tumorigenic cells could be enriched or depleted using markers but “non-tumorigenic” cells from the bottom of the hierarchy would always retain some tumorigenic capacity due to their ability to revert to tumorigenic states. E) A hierarchically organized cancer in which cells readily and reversibly interconvert between tumorigenic (red) and non-tumorigenic (yellow and green) states. Note that it would be difficult to experimentally distinguish case (E) from case (C). In cancers that are genetically unstable or subject to efficient reversible cell transitions, the cancer stem cell model may be untestable as it may be difficult to experimentally distinguish from cancers in which there is no hierarchy but where heterogeneity arises through clonal evolution.