Passenger mutations as a marker of clonal cell lineages in emerging neoplasia

Abstract

Cancer arises as the result of a natural selection process among cells of the body, favoring lineages bearing somatic mutations that bestow them with a proliferative advantage. Of the thousands of mutations within a tumor, only a small fraction functionally drive its growth; the vast majority are mere passengers of minimal biological consequence. Yet the presence of any mutation, independent of its role in facilitating proliferation, tags a cell's clonal descendants in a manner that allows them to be distinguished from unrelated cells. Such markers of cell lineage can be used to identify the abnormal proliferative signature of neoplastic clonal evolution, even at a stage which predates morphologically recognizable dysplasia. This article focuses on molecular techniques for assessing cellular clonality in humans with an emphasis on how they may be used for early detection of tumorigenic processes. We discuss historical as well as contemporary approaches and consider ways in which powerful new genomic technologies might be harnessed to develop a future generation of early cancer diagnostics.

Figure 1

Somatic mutations mark clonally derived populations. Every cell in the body has a unique genotype relative to the founding zygote as a result of random mutations (colored spots) that arise at low frequency during life. (A) Because conventional DNA sequencing of a tissue sample (blue circles) detects only the majority genotype (gray circles), most mutations will not be identified in normal tissues because they are present in only one or a few cells. If, however, a cell gains the ability to clonally proliferate and progeny remain spatially clustered, the unique mutational signature of the founder becomes manifest regardless of whether the cells are morphologically abnormal (B) or not (C). Screening for detectable mutations in non dysplastic, but at-risk tissues can be used to identify the abnormal proliferative signature of preneoplastic clones as a means detecting early cancer processes. Note that new random mutations arise in some cells during clonal expansion which are not detected. The mutations used to define a clone need not be the same ones functionally driving its expansion.

Figure 2

The unique features of mitochondrial mutations as a marker of cell lineage. To become detectable by routine sequencing techniques: (A) a mutant mitochondrial genome must (B) replicate and displace other genomes from its organelle and then (C) this mitochondria must outcompete or transform other mitochondria within its cell prior to (D) the marked cell clonally expanding within a tissue. (E) Dual color histochemical staining for functional loss of cytochrome c oxidase (blue coloration) can identify a subset of mutations in the mitochondrially-encoded COX1 gene for direct visualization of clonal populations in situ. Adapted from [75]. Panel E courtesy of Dr. Lawrence Loeb, University of Washington, Seattle.

Figure 3

Selected examples of important factors for consideration when interpreting clonality studies. See section 9 in the text for a detailed discussion of each scenario. Clusters of small circles in each panel represent a population of cells in a tissue. Each wedge represents a genomic site; gray and colored wedges respectively indicate wild-type and mutant genotypes. Blue boundaries indicate the true clonal relationship of cells derived from a neoplastic clonal expansion. Brown boundaries indicate an embryonically established clonal relationship. Dashed circles represent an area sampled for analysis. Boxes above each panel correspond to the genomic sites in the cell wedges and indicate the sample genotype as reported by a conventional method of sequencing that requires the majority of harvested cells to bear a mutation for it to be detectable. (A) Random mutations arising within individual cells in a tissue are not detectable by standard techniques. (B) If a cell bearing a unique mutation undergoes clonal expansion, it will become detectable when assessing this site. Note that new random mutations arise in some cells during clonal expansion which are not detected. (C) Clonal expansions will not be detectable if none of the genomic sites screened happen to be uniquely mutated in the founder cell. The probability of detecting a clone is a function of the number of sites screened and the frequency of random mutations in the founder cell. The latter, in turn, is dependent upon the per-cell-division mutation rate of each site and the number of cell divisions having occurred between the zygote and clone’s founding. (D) An elevated mutation frequency will facilitate a clone’s detection, although (E) in the absence of clonal expansion will have no effect on the detectable mutation load. (F) Clones that are small relative to the population sampled or that (G) become extensively mixed with other clones or (H) adjacent normal cells during expansion, are not detectable by conventional sequencing methods. (I) Based only on the presence of a detectable mutation, clonal patches arising during embryogenesis will be indistinguishable from those derived from neoplastic processes. Mutation spectrum, frequency and other contextual factors must be relied upon to infer the distinction.