Non-mutant genotypes in the surrounding tissue (Fig. 1A). If, however, a

Non-mutant genotypes in the surrounding tissue (Fig. 1A). If, however, a cell gains the ability to clonally proliferate as a result of one or more driver mutations, it will also carry along this larger number of neutral mutations to detectable level as chance passengers (Fig. 1B,C). Thus, the identification of any mutation in a tissue by conventional methods of aggregate DNA analysis, regardless of its functional status, is an indication that a clonal expansion has occurred. With few exceptions outside of the immune system, large clonal expansions arising in adults are abnormal and a signature of neoplasia. As a means of identifying preneoplastic clones, screening for neutral mutations has several advantages. First, the approach conceptually focuses on identifying the generic phenotype of abnormal growth patterns without the need for a priori knowledge of the heterogeneous genotypes that may induce it. The general concept can be easily transferred between cancer types having different characteristic drivers with little or no modification. Second, of all mutations carried by an emerging clone, a far greater number are passengers than drivers. Among the tens of thousands of mutations that have been identified in cancer genomes during the last three years, only a tiny subset is likely to be etiologically related [27,29,30]. Lastly, screening for mutations in strongly cancer-associated genes conceivably might even reduce the detectability of very early clones. Experimental introduction of powerful oncogenes, such as activated members of the ras pathway, induces growth arrest and senescence in otherwise untransformed human cells in vitro [31]. This suggests that the most primitive clones in vivo may be less likely to bear such lesions, having not yet accrued prior mutations to facilitate oncogene tolerance. The main drawback of relying on passenger mutations to identify clonal expansions is that they are not limited to a handful of defined loci, but occur scattered AZD-8835 chemical information throughout the genome. Mutagenesis, however, is not completelySemin Cancer Biol. Author manuscript; available in PMC 2011 October 15.Salk and HorwitzPagerandom; replication errors occur in certain hotspots orders of magnitude more frequently than elsewhere.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptIn this article we focus on techniques for identifying clones using neutral passengers and defer discussion of suspected driver-based methods to other excellent reviews [32?5]. For purposes herein, “neutral” is loosely defined as genetic or stable epigenetic changes identified based on methods not requiring knowledge of positively selectable loci. It is of course impossible to know with absolute certainty that mutation of a given site will not affect cell phenotype, but the spirit of the definition is to distinguish targeting of likely passengers from that of probable drivers. For the sake of brevity we limit the scope of our discussion to methods that are theoretically generalizable across multiple cell-types in the body. For example, we do not consider the elegant technique of using somatic rearrangement of Band T-cell Cynaroside web receptors as clonal markers in blood cancers [36], nor methods involving detection of random genomic integration sites of organ-specific viruses [37]. We begin with methods of historical importance involving X-chromosome inactivation before proceeding to approaches utilizing different varieties of mutational hotspots.4. X-linked genes and.Non-mutant genotypes in the surrounding tissue (Fig. 1A). If, however, a cell gains the ability to clonally proliferate as a result of one or more driver mutations, it will also carry along this larger number of neutral mutations to detectable level as chance passengers (Fig. 1B,C). Thus, the identification of any mutation in a tissue by conventional methods of aggregate DNA analysis, regardless of its functional status, is an indication that a clonal expansion has occurred. With few exceptions outside of the immune system, large clonal expansions arising in adults are abnormal and a signature of neoplasia. As a means of identifying preneoplastic clones, screening for neutral mutations has several advantages. First, the approach conceptually focuses on identifying the generic phenotype of abnormal growth patterns without the need for a priori knowledge of the heterogeneous genotypes that may induce it. The general concept can be easily transferred between cancer types having different characteristic drivers with little or no modification. Second, of all mutations carried by an emerging clone, a far greater number are passengers than drivers. Among the tens of thousands of mutations that have been identified in cancer genomes during the last three years, only a tiny subset is likely to be etiologically related [27,29,30]. Lastly, screening for mutations in strongly cancer-associated genes conceivably might even reduce the detectability of very early clones. Experimental introduction of powerful oncogenes, such as activated members of the ras pathway, induces growth arrest and senescence in otherwise untransformed human cells in vitro [31]. This suggests that the most primitive clones in vivo may be less likely to bear such lesions, having not yet accrued prior mutations to facilitate oncogene tolerance. The main drawback of relying on passenger mutations to identify clonal expansions is that they are not limited to a handful of defined loci, but occur scattered throughout the genome. Mutagenesis, however, is not completelySemin Cancer Biol. Author manuscript; available in PMC 2011 October 15.Salk and HorwitzPagerandom; replication errors occur in certain hotspots orders of magnitude more frequently than elsewhere.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptIn this article we focus on techniques for identifying clones using neutral passengers and defer discussion of suspected driver-based methods to other excellent reviews [32?5]. For purposes herein, “neutral” is loosely defined as genetic or stable epigenetic changes identified based on methods not requiring knowledge of positively selectable loci. It is of course impossible to know with absolute certainty that mutation of a given site will not affect cell phenotype, but the spirit of the definition is to distinguish targeting of likely passengers from that of probable drivers. For the sake of brevity we limit the scope of our discussion to methods that are theoretically generalizable across multiple cell-types in the body. For example, we do not consider the elegant technique of using somatic rearrangement of Band T-cell receptors as clonal markers in blood cancers [36], nor methods involving detection of random genomic integration sites of organ-specific viruses [37]. We begin with methods of historical importance involving X-chromosome inactivation before proceeding to approaches utilizing different varieties of mutational hotspots.4. X-linked genes and.