Volume 67, Issue 4, Pages 729-737 (April 2015) Intratumour Heterogeneity in Urologic Cancers: From Molecular Evidence to Clinical Implications  Marco Gerlinger, James W. Catto, Torben F. Orntoft, Francisco X. Real, Ellen C. Zwarthoff, Charles Swanton  European Urology  Volume 67, Issue 4, Pages 729-737 (April 2015) DOI: 10.1016/j.eururo.2014.04.014 Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 1 Sampling and sequencing approaches for the detection of genetic intratumour heterogeneity (ITH). (A) Cancer cell populations can be composed of multiple subclones, each defined by a different driver mutation profile. The presence of distinct driver mutations in the driver genes A–H is indicated in each cancer cell. Ubiquitous driver mutations are displayed in blue, those shared by several but not all clones in yellow, and mutations that are private to a single clone in red. Four tumour regions (R1–R4) are sampled for sequencing in this example. (B) Targeted sequencing (left column) can miss driver mutations and underreport heterogeneity. Standard depth exome sequencing (middle column) reveals the driver mutations present in the dominant clones in each sample but may miss rare subclonal drivers that become detectable through deep sequencing (right column). The subclones detected in each individual region differ, and subclones confined to tumour regions that were not sampled remain undetected in this example of a tumour with spatially separated subclones. (C) Phylogenetic tree reconstructed from all subclones shown in (A). Genes that acquired a driver mutation are indicated next to the branch where the mutation occurred. Ubiquitous mutations define the trunk of the phylogenetic tree, whereas shared and private mutations define the branches. (D) Phylogenetic trees inferred from different sequencing approaches differ in their level of detail. GL=germline DNA sequence. European Urology 2015 67, 729-737DOI: (10.1016/j.eururo.2014.04.014) Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 2 Branched evolution and driver mutation heterogeneity in clear cell renal cell carcinoma (ccRCC). Phylogenetic trees reconstructed from multiregion exome sequencing data from two ccRCC cases (RMH002 and RMH004) were adapted from Gerlinger et al. [14]. Genes that acquired a driver mutation are indicated next to the branch where the mutation occurred, and branch lengths are proportional to the number of somatic mutations. Parallel evolution through two distinct driver mutations in the PBRM1 gene and through driver mutations in the SMARCA4 and ARID1A genes that all encode parts of the SWI/SNF chromatin remodelling multiprotein complex was identified in RMH004. European Urology 2015 67, 729-737DOI: (10.1016/j.eururo.2014.04.014) Copyright © 2014 European Association of Urology Terms and Conditions

Fig. 3 Strategies incorporating intratumour heterogeneity (ITH) considerations into personalised medicine approaches. Assessment of genetic ITH in tumour tissues or in circulating tumour cells or cell-free DNA from the blood can reveal truncal or subclonal driver alterations as well as metrics quantifying the degree of ITH and signatures of genomic instability for correlation with patient outcomes. Predictions based on such novel biomarkers that take the subclonal genomic architecture and the evolutionary potential of heterogeneous tumours into account may be more accurate than traditional genetic biomarkers. Targeting of truncal drivers but also of “clinically dominant” drivers that determine the disease course even if they are subclonal, for example those fostering metastatic spread or rapid proliferation, and of genomic instability mechanisms may be effective therapeutic strategies in heterogeneous tumours. Combination therapy of multiple subclonal drivers may also be advantageous but could be restricted by toxicity. The genomic landscape of heterogeneous and evolving cancers is likely to change over time, and regular reassessments and adjustment of personalised therapies may be necessary. cfDNA=cell-free DNA; CTC=circulating tumour cell; ITH=intratumour heterogeneity. European Urology 2015 67, 729-737DOI: (10.1016/j.eururo.2014.04.014) Copyright © 2014 European Association of Urology Terms and Conditions