mutations in the prostate tumor genome, but based on our previous analysis of the THEMIS motif mutations in several genes in prostate cancer tissue (11), this genomic instability in prostate cancer may be the result of aberrant DNA repair in the prostate. In performing our genomic variation analysis, all of the variations found from the best aligned match in Genbank were scored, unless the number of variations represented >5% of the total insert length, in which case the clone sequence was rejected from the analysis (the level of genetic diversity among diﬀerent normal individuals rarely approaches 1% of the total sequence (17), so we reasoned that more than 5-fold higher would be unlikely even for the tumor DNA, and it probably represents a sequencing artifact (either on our side or the Genbank reference sequence). Using this criterion, ﬁve clones were rejected from the analysis of the tumor DNA, six from the normal prostate and seven from the blood DNA. All but one of the rejected clones represented non- unique sequence or included repeats (commonly centromeric DNA). The best alignment for the remaining clone is shown in Supplementary Figure 4, and may represent template-independent DNA synthesis performed by the tumor (18), or a fragment not yet sequenced by the various human genome projects. We favor the former explanation, since we were unable to PCR amplify this clone speciﬁc fragment from all normal unrelated DNA samples we examined (data not shown). Variants in repeats are more likely to be uncharac- terized polymorphisms (though not yet reported in dbSNP) and thus may increase the total count for both control and tumor DNAs. However, Supplementary Figure S5 demonstrates that the excess of tumor variations (especially substitutions) can not be merely explained by repeat variations. Thus we decided to include the repeat variants in our analysis. Subtracting the repeat variations from the total count changes the fre- quency estimate to 1.1 (±0.7) million somatic alterations per prostate tumor cell genome. The distribution of the variations by type (Supplementary Figure S5) shows that unlike normal prostate and blood, prostate tumors contain mostly substitutions, not deletions or insersions. However, the signiﬁcance of this ﬁnding is limited to small-scale tumor alterations, because the utilization of a four base cutter (AluI) in our cloning methodology does not allow us to adequately examine large-scale genomic instability (translocations, aneuploidy, etc.). The genomic analysis reported here demonstrates that normal prostate DNA does not show increased genomic instability compared to blood (Figure 1A), suggesting that ﬁxation artifacts (previously reported to artiﬁcially increase mutation rates in formalin ﬁxed samples; 19) do not play a major role in the data that we report here. The utilization of a PCR-free approach may be responsi- ble for this ﬁnding, since Taq DNA polymerase is thought to be the cause of these artifacts (19). Most of the observed variations in normal tissue (both prostate and blood) are probably yet-unidentiﬁed polymorphisms. Some of the tumor variations are also likely to be yet-unidentiﬁed polymorphisms. This is a major reason we subtracted the Nucleic Acids Research, 2009, Vol. 37, No. 22 7445 variation frequency of the normal from the tumor tissue, in order to measure the somatic variation frequency in tumors. Moreover, Figure 1B demonstrates that all patients show an increase in the variation frequency spe- ciﬁcally in the tumor genome (this trend is not signiﬁcant for individual patients, probably due to the low number of variations per patient). In summary, a PCR-free methodology that measures genomic variation rate reveals substantial somatic insta- bility in prostate cancer. This ﬁnding suggests at least a partial role for the mutator phenotype hypothesis (8) in prostate tumor progression. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Fabricio Rojas, Chris Haiman and Susan Roberts for technical support. We thank Laurence Kolonel and Brian Henderson for providing the tissue samples. FUNDING National Cancer Institute (P01 CA108964 project 1 to J.K.V.R.); the National Center for Research Resources (P20 RR020152-01 to N.M.M.) J.K.V.R. is a Medical Foundation Fellow at the University of Sydney. Funding for open access charge: National Institutes of Health grant. Conﬂict of interest statement. None declared. REFERENCES 1. Jemal,A., Siegel,R., Ward,E., Hao,Y., Xu,J., Murray,T. and Thun,M.J. (2008) Cancer statistics, 2008. CA Cancer J. Clin., 58, 71–96. 2. Bostwick,D.G., Burke,H.B., Djakiew,D., Euling,S., Ho,S.M., Landolph,J., Morrison,H., Sonawane,B., Shiﬄett,T., Waters,D.J. et al. (2004) Human prostate cancer risk factors. Cancer, 101, 2371–2490. 3. Marx,J. (2002) Debate surges over the origins of genomic defects in cancer. Science, 297, 544. 4. Greenman,C., Stephens,P., Smith,R., Dalgliesh,G.L., Hunter,C., Bignell,G., Davies,H., Teague,J., Butler,A., Stevens,C. et al. (2007) Patterns of somatic mutation in human cancer genomes. Nature, 446, 153–158. 5. Wood,L.D., Parsons,D.W., Jones,S., Lin,J., Sjo¨ blom,T., Leary,R.J., Shen,D., Boca,S.M., Barber,T., Ptak,J. et al. (2007) The genomic landscapes of human breast and colorectal cancers. Science, 318, 1108–1113. 6. The Cancer Genome Atlas Research Network. (2008) Comprehensive genomic characterization deﬁnes human glioblastoma genes and core pathways. Nature, 455, 1061–1068. 7. Stephens,P., Edkins,S., Davies,H., Greenman,C., Cox,C., Hunter,C., Bignell,G., Teague,J., Smith,R., Stevens,C. et al. (2005) A screen of the complete protein kinase gene family identiﬁes diverse patterns of somatic mutations in human breast cancer. Nat. Genet., 37, 590–592. 8. Loeb,L.A., Loeb,K.R. and Anderson,J.P. (2003) Multiple mutations and cancer. Proc. Natl Acad. Sci. USA, 100, 776–781. 9. Bielas,J.H., Loeb,K.R., Rubin,B.P., True,L.D. and Loeb,L.A. (2006) Human cancers express a mutator phenotype. Proc. Natl Acad. Sci. USA, 103, 18238–18242.