Deration when interpreting experimental findings. (i) Identification of a mutation in

Deration when interpreting experimental findings. (i) Identification of a mutation in a clonal population does not indicate causality Any mutation in a cell that undergoes clonal expansion will be passed onto progeny, irrespective of functional status (Fig. 3A,B). Given the size of the genome, more mutations carried by a neoplastic clone will be passengers than drivers [27, 29, 30]. To demonstrate driver status, supporting functional studies and/or repeated observations of a mutated loci in neoplastic clones from independent individuals are needed. (ii) Mutational marking of a clone is stochastic and not guaranteed A clone may exist and not be detected if none of the genomic sites being screened carry a unique mutation permitting it to be distinguished from the germline (Fig. 3C). The more sites examined, the higher the probability of detection becomes. Restricting a marker panel to suspected driver sites precludes detection of pathological clones driven by unknown factors. (iii) Elevated mutation rates facilitate clone detection but are not an absolute requirement Screening of mutational hotspots makes it practical with conventional technologies to have a reasonable probability of detecting a clone by random passenger mutations. Clones derived from a mutator lineage or cells residing in a highly mutagenic environment should be more densely marked with identifiable passengers (Fig. 3D), potentially reducing the number of markers needing to be interrogated to identify them. Emerging high-throughput sequencing technologies will eventually obviate the need to restrict screening to a fraction of the genome. (iv) Identification of one or more clonal mutations is not proof of genetic instability in the absence of collateral information Detection of a mutation requires clonal expansion with most traditional methods of aggregate DNA analysis. The probability of identifying clonal mutations in an expanded population is a function of the number of sites screened, the mutability of these sites and the number of cell divisions having occurred in the lineage leading up to the final expansion. Particularly when considering hotspots, mutations will be occasionally RR6 site detectable in any clonally-derived population at a statistically definable frequency. Because mutations are not routinely encountered in normal tissues, it is tempting to explain their presence in preneoplastic populations as the result of “genetic instability” (an elevated mutation rate). However, the phenomenon is more precisely explained by the fact that expansion does not routinely occur in most normal tissues. An elevated mutation rate itself will have no observable effect on clonal mutation Cycloheximide custom synthesis frequency in the absence of expansion (Fig. 3E). To determine mutation rate from a clonal mutation frequency it is necessary to (A) know that the population being assessed is clonal and (B) have some metric of the number of cell divisions that occurred in the period between the zygote and the founding of the final clonalSemin Cancer Biol. Author manuscript; available in PMC 2011 October 15.Salk and HorwitzPageoutgrowth. To infer that a rate is elevated, it is also necessary to know the normal in vivo mutation rate, which is, in turn, impossible to measure by this approach given that large clonal expansions do not routinely occur in normal adult tissue. Considering these complexities, the lack of resolution as to whether mutation rates are elevated in cancer is not surprising [1,8?1]. (v) Detectabili.Deration when interpreting experimental findings. (i) Identification of a mutation in a clonal population does not indicate causality Any mutation in a cell that undergoes clonal expansion will be passed onto progeny, irrespective of functional status (Fig. 3A,B). Given the size of the genome, more mutations carried by a neoplastic clone will be passengers than drivers [27, 29, 30]. To demonstrate driver status, supporting functional studies and/or repeated observations of a mutated loci in neoplastic clones from independent individuals are needed. (ii) Mutational marking of a clone is stochastic and not guaranteed A clone may exist and not be detected if none of the genomic sites being screened carry a unique mutation permitting it to be distinguished from the germline (Fig. 3C). The more sites examined, the higher the probability of detection becomes. Restricting a marker panel to suspected driver sites precludes detection of pathological clones driven by unknown factors. (iii) Elevated mutation rates facilitate clone detection but are not an absolute requirement Screening of mutational hotspots makes it practical with conventional technologies to have a reasonable probability of detecting a clone by random passenger mutations. Clones derived from a mutator lineage or cells residing in a highly mutagenic environment should be more densely marked with identifiable passengers (Fig. 3D), potentially reducing the number of markers needing to be interrogated to identify them. Emerging high-throughput sequencing technologies will eventually obviate the need to restrict screening to a fraction of the genome. (iv) Identification of one or more clonal mutations is not proof of genetic instability in the absence of collateral information Detection of a mutation requires clonal expansion with most traditional methods of aggregate DNA analysis. The probability of identifying clonal mutations in an expanded population is a function of the number of sites screened, the mutability of these sites and the number of cell divisions having occurred in the lineage leading up to the final expansion. Particularly when considering hotspots, mutations will be occasionally detectable in any clonally-derived population at a statistically definable frequency. Because mutations are not routinely encountered in normal tissues, it is tempting to explain their presence in preneoplastic populations as the result of “genetic instability” (an elevated mutation rate). However, the phenomenon is more precisely explained by the fact that expansion does not routinely occur in most normal tissues. An elevated mutation rate itself will have no observable effect on clonal mutation frequency in the absence of expansion (Fig. 3E). To determine mutation rate from a clonal mutation frequency it is necessary to (A) know that the population being assessed is clonal and (B) have some metric of the number of cell divisions that occurred in the period between the zygote and the founding of the final clonalSemin Cancer Biol. Author manuscript; available in PMC 2011 October 15.Salk and HorwitzPageoutgrowth. To infer that a rate is elevated, it is also necessary to know the normal in vivo mutation rate, which is, in turn, impossible to measure by this approach given that large clonal expansions do not routinely occur in normal adult tissue. Considering these complexities, the lack of resolution as to whether mutation rates are elevated in cancer is not surprising [1,8?1]. (v) Detectabili.