Recent studies have revealed extensive genetic diversity both between and within tumours. This heterogeneity affects key cancer pathways, driving phenotypic variation, and poses a significant challenge to personalized cancer medicine. A major cause of genetic heterogeneity in cancer is genomic instability. This instability leads to an increased mutation rate and can shape the evolution of the cancer genome through a plethora of mechanisms. By understanding these mechanisms we can gain insight into the common pathways of tumour evolution that could support the development of future therapeutic strategies.

Cancer chromosomal instability (CIN) results in an elevated rate of change of chromosome number and structure and generates intratumour heterogeneity1,2. CIN is observed in the majority of solid tumours and is associated with both poor prognosis and drug resistance3,4. Therefore, understanding a mechanistic basis for CIN is paramount. Here we find evidence for impaired replication fork progression and elevated DNA replication stress in CIN+ colorectal cancer (CRC) cells relative to CIN− CRC cells, with structural chromosome abnormalities precipitating chromosome missegregation in mitosis. We identify three novel CIN-suppressor genes (PIGN (MCD4), RKHD2 (MEX3C) and ZNF516 (KIAA0222)) encoded on chromosome 18q, which is subject to frequent copy number loss in CIN+ CRC. 18q loss was temporally associated with aneuploidy onset at the adenoma-carcinoma transition. CIN-suppressor gene silencing leads to DNA replication stress, structural chromosome abnormalities and chromosome missegregation. Supplementing cells with nucleosides, to alleviate replication-associated damage5, reduces the frequency of chromosome segregation errors following CIN-suppressor gene silencing and attenuates segregation errors and DNA damage in CIN+ cells. These data implicate a central role for replication stress in the generation of structural and numerical CIN, which may inform new therapeutic approaches to limit intratumour heterogeneity.

The contribution of whole-genome doubling to chromosomal instability (CIN) and tumor evolution is unclear. We use long-term culture of isogenic tetraploid cells from a stable diploid colon cancer progenitor to investigate how a genome-doubling event affects genome stability over time. Rare cells that survive genome doubling demonstrate increased tolerance to chromosome aberrations. Tetraploid cells do not exhibit increased frequencies of structural or numerical CIN per chromosome. However, the tolerant phenotype in tetraploid cells, coupled with a doubling of chromosome aberrations per cell, allows chromosome abnormalities to evolve specifi cally in tetraploids, recapitulating chromosomal changes in genomically complex colorectal tumors. Finally, a genome-doubling event is independently predictive of poor relapse-free survival in early-stage disease in two independent cohorts in multivariate analyses [discovery data: hazard ratio (HR), 4.70, 95% confi dence interval (CI), 1.04-21.37; validation data: HR, 1.59, 95% CI, 1.05-2.42]. These data highlight an important role for the tolerance of genome doubling in driving cancer genome evolution. SIGNIFICANCE:Our work sheds light on the importance of whole-genome-doubling events in colorectal cancer evolution. We show that tetraploid cells undergo rapid genomic changes and recapitulate the genetic alterations seen in chromosomally unstable tumors. Furthermore, we demonstrate that a genome-doubling event is prognostic of poor relapse-free survival in this disease type. Cancer Discov; 4(2);

Chromosomal instability (CIN)-which is a high rate of loss or gain of whole or parts of chromosomes-is a characteristic of most human cancers and a cause of tumour aneuploidy and intra-tumour heterogeneity. CIN is associated with poor patient outcome and drug resistance, which could be mediated by evolutionary adaptation fostered by intra-tumour heterogeneity. In this review, we discuss the clinical consequences of CIN and the challenges inherent to its measurement in tumour specimens. The relationship between CIN and prognosis supports assessment of CIN status in the clinical setting and suggests that stratifying tumours according to levels of CIN could facilitate clinical risk assessment.

Cancer drug resistance is a major problem, with the majority of patients with metastatic disease ultimately developing multidrug resistance and succumbing to their disease. Our understanding of molecular events underpinning treatment failure has been enhanced by new genomic technologies and pre‐clinical studies. Intratumour genetic heterogeneity (ITH) is a prominent contributor to therapeutic failure, and it is becoming increasingly apparent that individual tumours may achieve resistance via multiple routes simultaneously – termed polyclonal resistance. Efforts to target single resistance mechanisms to overcome therapeutic failure may therefore yield only limited success. Clinical studies with sequential analysis of tumour material are needed to enhance our understanding of inter‐clonal functional relationships and tumour evolution during therapy, and to improve drug development strategies in cancer medicine.

Background Chromosomal instability (CIN) is thought to be associated with poor prognosis in solid tumours, however, evidence from pre-clinical and mouse tumour models suggest that CIN may paradoxically enhance or impair cancer cell fitness. Breast cancer prognostic expression signature sets, which reflect tumour CIN status, efficiently delineate outcome in ER-positive breast cancer in contrast to ER-negative breast cancer, suggesting that the relationship of CIN with prognosis differs in these two breast cancer subtypes. Methods Direct assessment of CIN requires single cell analysis methods such as centromeric fluorescence in situ hybridisation (FISH) aimed at determining the variation around the modal number of two or more chromosomes within individual tumour nuclei. Here we document the frequency of tumour CIN by dual centromeric FISH analysis in a retrospective primary breast cancer cohort of 246 patients with survival outcome. Results There was increased CIN and clonal heterogeneity in ER-negative compared to ER-positive breast cancer. Consistent with a negative impact of CIN on cellular fitness, extreme CIN in ER-negative breast cancer was an independent variable associated with improved long-term survival in multivariate analysis. In contrast, a linear relationship of increasing CIN with poorer prognosis in ER-positive breast cancer was observed, using three independent measures of CIN. Conclusions The paradoxical relationship between extreme CIN and cancer outcome in the ER-negative cohorts may explain why prognostic expression signatures, reflecting tumour CIN status, fail to predict outcome in this subgroup. Impact Assessment of tumour CIN status may support risk stratification in ER negative breast cancer and requires prospective validation.

Subclonal cancer populations change spatially and temporally during the disease course. Studies are revealing branched evolutionary cancer growth with low-frequency driver events present in subpopulations of cells, providing escape mechanisms for targeted therapeutic approaches. Despite such complexity, evidence is emerging for parallel evolution of subclones, mediated through distinct somatic events converging on the same gene, signal transduction pathway, or protein complex in different subclones within the same tumor. Tumors may follow gradualist paths (microevolution) as well as major shifts in evolutionary trajectories (macroevolution). Although macroevolution has been subject to considerable controversy in post-Darwinian evolutionary theory, we review evidence that such nongradual, saltatory leaps, driven through chromosomal rearrangements or genome doubling, may be particularly relevant to tumor evolution. Adapting cancer care to the challenges imposed by tumor micro- and macroevolution and developing deeper insight into parallel evolutionary events may prove central to improving outcome and reducing drug development costs.

Defining mechanisms that generate intratumour heterogeneity and branched evolution may inspire novel therapeutic approaches to limit tumour diversity and adaptation. SETD2 (Su(var), Enhancer of zeste, Trithorax-domain containing 2) trimethylates histone-3 lysine-36 (H3K36me3) at sites of active transcription and is mutated in diverse tumour types, including clear cell renal carcinomas (ccRCCs). Distinct SETD2 mutations have been identified in spatially separated regions in ccRCC, indicative of intratumour heterogeneity. In this study, we have addressed the consequences of SETD2 loss-of-function through an integrated bioinformatics and functional genomics approach. We find that bi-allelic SETD2 aberrations are not associated with microsatellite instability in ccRCC. SETD2 depletion in ccRCC cells revealed aberrant and reduced nucleosome compaction and chromatin association of the key replication proteins minichromosome maintenance complex component (MCM7) and DNA polymerase δ hindering replication fork progression, and failure to load lens epithelium-derived growth factor and the Rad51 homologous recombination repair factor at DNA breaks. Consistent with these data, we observe chromosomal breakpoint locations are biased away from H3K36me3 sites in SETD2 wild-type ccRCCs relative to tumours with bi-allelic SETD2 aberrations and that H3K36me3-negative ccRCCs display elevated DNA damage in vivo. These data suggest a role for SETD2 in maintaining genome integrity through nucleosome stabilization, suppression of replication stress and the coordination of DNA repair.