Many years ago, I put forth the concept that the mutation rate of nonmalignant cells is insufficient to generate the large numbers of mutations that are present in human cancers1. Instead, it was hypothesized that cancers express a mutator phenotype, and as a result progressively accumulate2 large numbers of mutations during tumour progression. The human genome is dynamic it is estimated that each cell undergoes 20,000 DNA damaging events3���5 and 10,000 replication errors per cell per day6. As a result, mutations occur throughout the genome, including in genes that maintain genetic stability. DNA damage that escapes correction by base excision repair (BER) or nucleotide excision repair (NER)4 can generate misincorporations during DNA replication7. Misincorporations by mutant DNA polymerases5���7 that escape mismatch repair (MMR)8 result in single-base substi- tutions. Unrepaired DNA alterations and crosslinks that block DNA replication can result in chromosome rearrangements, amplifications and deletions9. The number of proteins that function in DNA replica- tive processes in human cells is not known. However, studies in yeast indicate that 100 genes are required for the maintenance of genetic stability10. Among these are genes that encode error-prone DNA polymer- ases that can replicate past bulky lesions on DNA11. Mutations or misregulation of any of these genes could increase the probability that subsequent mutations will occur in oncogenes (resulting in driver mutations that confer a growth advantage). Such repetitive cycles of mutagenesis and selection mimic Darwinian evolution. Most mutations are ���passengers��� that do not confer a growth advantage. The concept of cancer being initiated by DNA damage and the generation of large numbers of driver, mutator and passenger mutations after each round of selection is illustrated in FIG.��1. In addition to driver mutations, there are subclonal mutations that are present in a large proportion of cells as well as random mutations that are generated during the last round of clonal selection. By the time a solid tumour is detected, it frequently measures 1 cm3 and encompasses 108���109 cells, each cell containing tens of thousands of clonal, subclonal and random mutations12. In order for environmental agents to introduce mutations that cause cancer, the mutations would need to be in excess of those produced by normal cellular pro cesses. The major source of endogenous DNA damage is likely to be reactive oxygen spe- cies (ROS) and related reactive molecules13. The principal alteration produced by ROS is 8-oxo-deoxyguanosine (8-oxo-dG)13, and mice harbouring mutations in genes that encode proteins that repair oxygen-damaged DNA are cancer-prone4. DNA damage by ROS14 as well as errors by replicative DNA polymerases in��vitro2,15 and methylcytidine deamination16 can result in a dispropor- tionally high frequency of single GC���AT transitions. These are also the most fre- quent mutations that accumulate in human tumours17. Thus, it is tempting to speculate that these processes are a major source of mutations in spontaneous��tumours. Evidence for the expression of a mutator phenotype in human cancer has been presented18,19. Recent studies in mice have shown that if the genes encoding the repli- cative DNA polymerases Pol �� or Pol �� are replaced with genes harbouring mutations that render them error-prone, tumours occur in various tissues20 this lends further credence to the mutator phenotype concept (BOX��1). The efficiency by which cancers arise with and without mutator mutations has also been modelled by varying all clinically relevant parameters21. The importance of a mutator mutation is greatest when more oncogenic mutations are required for the commitment to cancer. For cancers that require only one or two mutations, such as inherited retinoblastoma, a mutator pheno- type may not be necessary. However, for most cancers that require three or more driver mutations, a mutator phenotype may be inevitable21. Based on age of onset, it is postulated that prostate cancer, for example, requires as many as 12 driver mutations117,118. By contrast, I have argued that human can- cers contain thousands of mutations, many of which are random, and that mutations in multiple pathways can result in a malignant phenotype. This Perspective focuses on results obtained from DNA sequencing, and the implications of a mutator phenotype in can- cer. The feasibility of modifying the growth of cancers by altering the rate of accumulation of mutations is considered. Undoubtedly, changes at the level of transcription and trans- lation also contribute to a mutator phenotype in cancer. However, these epigenetic changes will not be considered owing to limited direct evidence that they are stably transferred from one generation to another in cancer cells, their reversible nature and the lack of knowledge about the functional significance of specific alterations. GENOMIC INSTABILITY IN CANCER ��� OPINION Human cancers express mutator phenotypes: origin, consequences and targeting Lawrence A.��Loeb Abstract | Recent data on DNA sequencing of human tumours have established that cancer cells contain thousands of mutations. These data support the concept that cancer cells express a mutator phenotype. This Perspective considers the evidence supporting the mutator phenotype hypothesis, the origin and consequences of a mutator phenotype, the implications for personalized medicine and the feasibility of ablating tumours by error catastrophe. PERSPECTIVES 450 | JUNE 2011 | VOLUME 11 www.nature.com/reviews/cancer �� 2011 Macmillan Publishers Limited. All rights reserved

Nature Reviews | Cancerare Environmental sources Endogenous sources DNA damage Mutation Mutation in mutator genes Repetitive selection for mutants and mutators Selection for malignant phenotypes Clinically detectable cancer DNA repair Nutrition Angiogenesis Hypoxia Sequencing of human tumour DNA Ironically, the most significant evidence for the existence of a mutator phenotype comes from The Cancer Genome Atlas, which was designed to catalogue mutations in human cancers with the expectation of identifying new targets for chemotherapy. The results indicate that each tumour is unique and contains tens to hundreds of thousands of mutations17,18. It is important to emphasize that the current methods of DNA sequencing detect only the most frequent substitu- tions at each position a single base change is detected only if present in 10% of the molecules. To detect less frequent substi- tutions, it is necessary to sequence either the same region multiple times (deep sequencing) or to sequence single mol- ecules without errors. Thus, mutations catalogued in The Cancer Genome Atlas and similar cancer databanks are not derived from deep sequencing and are therefore predominately clonal sub- clonal22,23 and random23,24 mutations have not been extensively characterized. Moreover, the nucleotide sequences of repeats and telomeres have not yet been determined owing to slippage of DNA polymerase during PCR amplification, the lack of fixed primer sites and com- plexities in aligning reads. Repetitive DNA sequences often assume non-B DNA con- formations that are frequently mutated25,26. Also, deletions, insertions and rearrange- ments are frequently not reported. As a result, no human genome has yet been completely sequenced. Exon sequencing. A compilation of the numbers and types of mutations found in exons from a variety of tumours is presented in TABLE��1. More than 1,000 different genes have been reported to be mutated in human tumours, and many tumours contain as many as 100 non-synonymous mutations. In any tumour type, none of the genes is invariably mutated nor is there a set of mutated genes that are diagnostic of a spe- cific tumour17. Next-generation sequencing has identified many known cancer genes, including TP53, KRAS and epidermal growth factor receptor (EGFR)27, as well as genes that previously were not known to be involved in carcinogenesis. Many genes contain multiple mutations. A single breast tumour, for exam- ple, contained 70 mutant protein kinases some with three separate substitutions28. The most frequently reported mutant gene was TP53, and in lung tumours TP53 mutations correlated with tumour grade: for example, somatic mutations in TP53 were reported in 13%, 24% and 52% of tumours of grades 1, 2 and 5, respectively29. So far, only a few new genes have been shown to be commonly mutated, and these are neither highly prevalent nor in multiple tumour��types. Whole-genome sequencing. The types of somatic mutations in normal human tissue have been difficult to establish. However, DNA sequences of family members, gen- erations apart, indicate that single-base transitions are the most common mutations detected30. Most mutations reported in tumours (TABLE��1) are also single-base substi- tutions CG���TA transitions predominate. In lung tumour cell lines23,31 and melanoma cell lines31, the mutation frequency on the tran- scribed strand is lower than that on the non-transcribed strand, which affirms the concept of preferential removal of endo- genous DNA damage by transcription- coupled NER32. In some tumours, the range of mutations is unique and is indicative of expo- sure to environmental agents. Tobacco smoke contains large amounts of polycyclic hydro- carbons and aromatic amines33 that form bulky adducts in DNA when bypassed by a translesion DNA polymerase (Pol ��)34, they result in predominantly G���T trans versions, which are precisely the most frequent errors reported in lung cancers29,35,36. In skin cancer, the most frequent mutations are found at potential sites of ultraviolet-radiation-induced pyrimidine dimer formation23,27. Thus, DNA sequencing will increasingly yield impor- tant clues about environmental exposures to mutagens that could enhance a mutator phenotype. Even in haematopoietic malignancies in which morphological homogeneity is fre- quently diagnostic, genetic heterogeneity is extensive. For example, the most successful demonstration of targeted cancer therapy is the treatment of myelogenous leukaemia with imatinib37,38, a specific inhibitor of the breakpoint cluster region (BCR)���ABL1 fusion kinase. Resistance emerges in more than 30% of patients and is most frequently mediated by a point mutation in the ATP binding site of ABL1 (REF. 39). The emergence of resistance is associated with pre-existing mutations40. In one study, whole-genome sequencing revealed only Figure 1 | Cascade of mutations during tumour progression. In the case of solid tumours, epidemiological evidence indicates that as many as 20 years pass between the time an individual is exposed to a carcinogen to the clinical appearance of a tumour. Various barriers to tumour progression exist, including DNA repair processes, the availability of nutrition, the requirement of angiogenesis to allow the tumour to increase in size and responses to hypoxia. Circles represent mutations in genes that result in enhanced mutagenesis, triangles indicate driver mutations that selected on the basis of changes in the tumour microenvironment and white rectangles represent passenger mutations. PERSPECTIVES NATURE REVIEWS | CANCER VOLUME 11 | JUNE 2011 | 451 �� 2011 Macmillan Publishers Limited. All rights reserved