The conceptual foundation dates back to 1976, when Peter Nowell proposed that tumour cell populations evolve through acquired genetic lability, with stepwise selection of variant sublines driving progression. Half a century of bulk sequencing vindicated the model, revealing branched evolution, intratumoural heterogeneity and the selective sweeps that follow treatment. Yet bulk measurements average across millions of cells, obscuring which mutation resides in which cell and, crucially, what that mutation actually does to the cell carrying it. The review’s authors argue that closing this genotype-to-phenotype gap is now the central task of cancer evolutionary biology.

One striking motivation comes from healthy tissue. Landmark studies of normal skin, oesophagus, colon, endometrium, bladder and bronchial epithelium have shown that somatic mutations in canonical cancer driver genes are under pervasive positive selection in tissue that looks entirely normal under the microscope. In sun-exposed skin, roughly a quarter of cells carry cancer-associated mutations, and mutation burdens in some cells rival those of tumours. Clonal haematopoiesis, the age-related expansion of blood cells carrying mutations in genes such as DNMT3A, TET2 and JAK2, likewise demonstrates that genetic mosaicism is a feature of ordinary physiology and ageing, seeding the pre-malignant landscape from which frank cancers emerge.

Mapping this diversity, however, is only the first step. The review emphasises that understanding somatic clonal evolution requires defining the phenotypes that give mutated clones a fitness advantage, whether those phenotypes involve altered differentiation, survival, proliferation or interaction with the microenvironment. This is where genotype-aware single-cell multi-omics enters. Methods such as G&T-seq and its descendants physically split or barcode the genome and transcriptome of an individual cell, while genotyping-of-transcriptomes approaches recover expressed mutations directly from single-cell RNA-sequencing data. Targeted strategies enrich for known mutant loci, and chromatin-focused assays now co-capture mutations alongside single-cell accessibility profiles.

The biological payoffs have been substantial. In clonal haematopoiesis, single-cell multi-omics has shown that the effects of a mutation are often cell-state specific. DNMT3A R882 mutations, for example, were found to perturb early progenitor states through selective hypomethylation, a phenotype invisible to bulk assays. Splicing aberrations in haematopoietic clonal outgrowths display distinct cell-type-specific impacts, and maps linking genotypes to chromatin accessibility profiles reveal how individual mutations reshape regulatory landscapes in a lineage-dependent manner. In myeloproliferative neoplasms, clonally resolved analyses have traced how JAK2 and CALR mutations propagate through differentiation hierarchies, while work in acute myeloid leukaemia has connected RAS-mutant leukaemia stem cells to clinical resistance against the BCL-2 inhibitor venetoclax.

Beyond single time points, the review highlights the power of coupling phylogenetic reconstruction with phenotypic measurement. Endogenous marks such as somatic point mutations, copy-number alterations, mitochondrial DNA mutations, microsatellite shifts and stochastic epimutations each leave heritable traces that allow researchers to infer the ancestral relationships among single cells. Mitochondrial mutations in particular have enabled lineage tracing directly in human samples, and somatic epimutations have recently been used to chart the dynamics of blood ageing. Reconstructed single-cell phylogenies can then be time-calibrated, converting a branching diagram into a chronogram that estimates when a clone originated within a patient’s lifespan.

Such temporal mapping demands careful statistical treatment. The authors describe phylogenetic frameworks that quantify heritability and plasticity of cell states, decoupling genetic inheritance from non-genetic, environmentally driven transitions. Molecular clock models, whether strict or relaxed, permit inference of mutation rates and timing of clonal expansions, and phylodynamic approaches borrowed from pathogen genetics now illuminate how tumour population sizes fluctuate over the course of disease. Applied to colorectal cancer, these tools have revisited the Big Bang model of tumour growth, in which most subclonal diversity is generated in an early expansion rather than through later selective sweeps, and have documented phenotypic plasticity under genetic control during malignant progression.

Spatial context adds a further dimension. Multiclonal invasion patterns in breast tumours, spatially resolved copy-number maps in benign and malignant tissue, and spatial genomics of cancer clones all demonstrate that evolutionary dynamics are constrained by tumour architecture. Mechanical confinement has been shown to govern phenotypic plasticity in melanoma, and harsh microenvironments select for glycolytic phenotypes in early breast cancer. Integrating spatially resolved or lineage-resolved phenotypes with genotype maps is therefore revealing how selection operates not just on mutations but on the cell states and niches in which those mutations find themselves.

Translationally, the authors argue that genotype-to-phenotype mapping can expose therapeutic vulnerabilities for the precision elimination of disease-propagating mutant cells. Because mutant phenotypes are often confined to specific cell states or lineages, vulnerabilities may exist that spare wild-type tissue. Single-cell analyses have identified drug-tolerant persister states, non-genetic determinants of clonal fitness, and epigenetically inherited plasticity that drives drug resistance through one-to-many genotype-to-phenotype relationships. In glioblastoma, recurring cellular states whose abundance is modulated by genetic aberrations suggest combination strategies; in IDH-mutant oligodendroglioma, mutant IDH inhibitors induce lineage differentiation detectable at single-cell resolution. Evolutionary steering, in which treatment is designed to guide tumours toward collateral sensitivities, becomes more tractable when clonal phenotypes can be read directly in patients.

The review closes with a sober assessment of remaining challenges. Whole-genome amplification artefacts, allelic dropout and tissue dissociation biases still limit sensitivity and fidelity, and artifacts in mitochondrial DNA analyses can misinform phylogenetic inference if uncorrected. Computational methods continue to mature, from probabilistic single-cell phylogeny inference that accounts for sequencing error to models that relax the infinite-sites assumption when back mutations and parallel evolution occur. Yet the trajectory is clear: by pairing genotype with phenotype in the same cell and embedding those pairs within time-calibrated phylogenies, researchers can now define, directly in primary human samples, the mechanisms underlying clonal expansion in both healthy and malignant tissues. For a disease that evolves to evade every therapy thrown at it, that may prove the most consequential lens oncology has yet acquired.

Subject of Research: Single-cell multi-omics mapping of genotype and phenotype co-evolution in clonal evolution of healthy and cancerous tissues

Article Title: A single-cell lens into the co-evolution of genotypes and phenotypes in cancer

Article References: Izzo, F., Prieto, T., Potenski, C., & Landau, D. A. (2026). A single-cell lens into the co-evolution of genotypes and phenotypes in cancer. Nature Reviews Cancer. https://doi.org/10.1038/s41568-026-00970-8

Image Credits: AI Generated

DOI: 10.1038/s41568-026-00970-8

Keywords: single-cell multi-omics, clonal evolution, genotype-phenotype mapping, tumour heterogeneity, clonal haematopoiesis, single-cell phylogenetics, lineage tracing, somatic mosaicism, phenotypic plasticity, cancer genomics, epigenetics, therapeutic vulnerabilities

Extrachromosomal DNA, circular DNA molecules separate from the chromosomes, are notorious for harboring oncogenes that fuel cancer progression. Unlike chromosomal DNA, ecDNAs replicate and segregate imperfectly during cell division, often resulting in their rapid loss from daughter cells. Until now, the processes that ensure ecDNA retention and amplification despite this challenge remained poorly understood, limiting our grasp of cancer biology at a genomic level.

The team employed advanced evolutionary simulations to dissect the interplay between ecDNA retention fidelity and natural selection in the context of tumor cell populations. Their models revealed that ecDNAs could only achieve significant amplification when the fidelity of their retention during mitosis exceeded 90%. If retention rates dropped below this threshold, even potent selective advantages conferred by oncogenes failed to stabilize ecDNA presence, highlighting that near-perfect mitotic retention is essential for ecDNA-driven oncogenic expansion.

Intriguingly, this theoretical minimum retention rate mirrored experimental observations garnered through cutting-edge live-cell imaging. A single retention element embedded within ecDNAs was sufficient to confer a roughly 10% failure rate per mitosis, confirming the simulation predictions. This tight correlation underscores the biological importance of retention elements in sustaining the oncogenic functions of ecDNA lineages within tumors.

Further analyses of patient tumor samples revealed that nearly all ecDNA amplicons containing oncogenes also carried retention elements, with 98% co-amplification observed. These retention elements frequently co-localized with oncogenes on large ecDNA segments often exceeding one megabase in size, vastly larger than the oncogene sequences themselves. This excess DNA likely harbors multiple retention elements, collectively enhancing mitotic stability and promoting persistent oncogene expression.

Contrastingly, linear chromosomal amplifications displayed more variable sizes and a sparser distribution of retention elements, suggesting a fundamental difference in how ecDNA and chromosomal amplifications evolve and maintain themselves in cancer cells. DNA segments lacking retention elements were commonly linked to those with retention elements on ecDNAs, but such associations were absent in linear amplifications, reinforcing the specific structural significance of retention elements for extrachromosomal maintenance.

Investigating spatial patterns, the study found that the local density of retention elements inversely correlated with ecDNA amplicon size. Genomic regions rich in retention elements tended to give rise to smaller ecDNA circles, whereas low-density areas favored larger ecDNA amplicons to encompass at least one retention element. This nuanced relationship influences the architecture of ecDNA and indicates that cancer cells exploit retention element distribution to optimize oncogene amplification efficiently.

Beyond tumor contexts, the researchers also explored the presence of retention elements in smaller, nonclonal extrachromosomal circular DNAs—known as microDNAs—which are prevalent in normal somatic tissues but typically not amplified. Remarkably, although most microDNAs lacked retention elements, there was a significant enrichment of these elements within microDNAs compared to random genomic segments across diverse human cell lines, implicating retention elements even in the persistence of small circular DNAs outside cancerous settings.

Epigenetic profiling of retention elements demonstrated lower DNA methylation levels compared to matched genomic intervals, suggesting a unique chromatin environment that might favor retention element function. Targeted methylation of retention elements using CRISPRoff technology reduced ecDNA tethering within cells, highlighting the critical role of their epigenetic state in maintaining ecDNA stability.

Functionally, these findings converge on a model in which retention elements serve as molecular anchors securing ecDNA during mitosis, thereby enhancing their inheritance and enabling sustained oncogene-driven proliferation. This synergy between retention and selection fundamentally shapes ecDNA-driven tumor evolution and offers promising targets for disrupting the oncogenic potential of extrachromosomal genetic material.

The implications are profound: targeting retention elements or their associated molecular machinery could destabilize ecDNA maintenance, leading to loss of oncogene amplification and potentially sensitizing tumors to existing therapies. This strategy opens a new frontier in cancer treatment, focused on extrachromosomal genetic regulation rather than chromosomal mutations alone.

In conclusion, this study elucidates how genetic retention elements are central to the selective amplification and persistence of oncogene-containing ecDNAs in cancer. By bridging computational modeling, patient-derived genomic data, and epigenetic analyses, the work paints a comprehensive picture of extrachromosomal DNA biology with far-reaching consequences for cancer research and therapy development.

Subject of Research:
Retention elements that facilitate the maintenance and selective amplification of oncogene-containing extrachromosomal DNA in cancer cells.

Article Title:
Genetic elements promote retention of extrachromosomal DNA in cancer cells.

Article References:
Sankar, V., Hung, K.L., Gnanasekar, A. et al. Genetic elements promote retention of extrachromosomal DNA in cancer cells. Nature (2025). https://doi.org/10.1038/s41586-025-09764-8

DOI:
https://doi.org/10.1038/s41586-025-09764-8