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A fundamental goal in cancer research is to understand the mechanisms of cell transformation. This is key to developing more efficient cancer detection methods and therapeutic approaches. One milestone towards this objective is the identification of all the genes with mutations capable of driving tumours. Since the 1970s, the list of cancer genes has been growing steadily. Because cancer driver genes are under positive selection in tumorigenesis, their observed patterns of somatic mutations across tumours in a cohort deviate from those expected from neutral mutagenesis. These deviations, which constitute signals of positive selection, may be detected by carefully designed bioinformatics methods, which have become the state of the art in the identification of driver genes. A systematic approach combining several of these signals could lead to a compendium of mutational cancer genes. In this Review, we present the Integrative OncoGenomics (IntOGen) pipeline, an implementation of such an approach to obtain the compendium of mutational cancer drivers. Its application to somatic mutations of more than 28,000 tumours of 66 cancer types reveals 568 cancer genes and points towards their mechanisms of tumorigenesis. The application of this approach to the ever-growing datasets of somatic tumour mutations will support the continuous refinement of our knowledge of the genetic basis of cancer.This Review provides a brief historical perspective of our understanding of the role of cancer genes before presenting the Integrative OncoGenomics (IntOGen) platform, a bioinformatics method of mutational driver identification, which is beginning to reveal the compendium of driver genes across many tumour types as well as alluding to their tumorigenic mechanisms.

An algorithm that uses evolutionary data to predict disease variants makes a case for embracing computational evidence for clinical interpretation of genetic variation.

Colorectal carcinoma (CRC) is a common cause of mortality 1 , but a comprehensive description of its genomic landscape is lacking 2 – 9 . Here we perform whole-genome sequencing of 2,023 CRC samples from participants in the UK 100,000 Genomes Project, thereby providing a highly detailed somatic mutational landscape of this cancer. Integrated analyses identify more than 250 putative CRC driver genes, many not previously implicated in CRC or other cancers, including several recurrent changes outside the coding genome. We extend the molecular pathways involved in CRC development, define four new common subgroups of microsatellite-stable CRC based on genomic features and show that these groups have independent prognostic associations. We also characterize several rare molecular CRC subgroups, some with potential clinical relevance, including cancers with both microsatellite and chromosomal instability. We demonstrate a spectrum of mutational profiles across the colorectum, which reflect aetiological differences. These include the role of Escherichia coli pks+ colibactin in rectal cancers 10 and the importance of the SBS93 signature 11 – 13 , which suggests that diet or smoking is a risk factor. Immune-escape driver mutations 14 are near-ubiquitous in hypermutant tumours and occur in about half of microsatellite-stable CRCs, often in the form of HLA copy number changes. Many driver mutations are actionable, including those associated with rare subgroups (for example, BRCA1 and IDH1 ), highlighting the role of whole-genome sequencing in optimizing patient care. Whole-genome sequencing of more than 2,000 colorectal carcinoma samples provides a highly detailed view of the genomic landscape of this cancer and identifies new driver mutations.

DNA base damage is a major source of oncogenic mutations 1 . Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation 2 . Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication 3 , 4 , we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts 5 . The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution. How strand-asymmetric processes such as replication and transcription interact with DNA damage to drive mechanisms of repair and mutagenesis is explored.

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion 1 , 2 . Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes. Mutagenic lesions such as those that give rise to cancer frequently segregate—unrepaired—during cell division, resulting in phasing of multiple alleles across generations of daughter cells and consequent tumour heterogeneity.

Lymphocyte activation is associated with rapid increase of both the glycolytic activator fructose 2,6-bisphosphate (Fru-2,6-P 2 ) and the enzyme responsible for its synthesis, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFKFB3 gene, which encodes for the most abundant PFK-2 isoenzyme in proliferating tissues, has been found overexpressed during cell activation in several models, including immune cells. However, there is limited knowledge on the pathways underlying PFKFB3 regulation in human T-lymphocytes, and the role of this gene in human immune response. The aim of this work is to elucidate the molecular mechanisms of PFKFB3 induction during human T-lymphocyte activation by mitotic agents. The results obtained showed PFKFB3 induction during human T-lymphocyte activation by mitogens such as phytohemagglutinin (PHA). PFKFB3 increase occurred concomitantly with GLUT-1, HK-II, and PCNA upregulation, showing that mitotic agents induce a metabolic reprograming process that is required for T-cell proliferation. PI3K–Akt pathway inhibitors, Akti-1/2 and LY294002, reduced PFKFB3 gene induction by PHA, as well as Fru-2,6-P 2 and lactate production. Moreover, both inhibitors blocked activation and proliferation in response to PHA, showing the importance of PI3K/Akt signaling pathway in the antigen response of T-lymphocytes. These results provide a link between metabolism and T-cell antigen receptor signaling in human lymphocyte biology that can help to better understand the importance of modulating both pathways to target complex diseases involving the activation of the immune system.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A*01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote \"off-the-shelf\" precision immunotherapies, alleviating limitations of personalized treatments.

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.

Neoantigens are now recognized drivers of the antitumor immune response. Recurrent neoantigens, shared among groups of patients, have thus become increasingly coveted therapeutic targets. Here, we report on the data-driven identification of a robustly presented, immunogenic neoantigen that is derived from the combination of HLA-A·01:01 and RAS.Q61K. Analysis of large patient cohorts indicated that this combination applies to 3% of patients with melanoma. Using HLA peptidomics, we were able to demonstrate robust endogenous presentation of the neoantigen in 10 tumor samples. We detected specific reactivity to the mutated peptide within tumor-infiltrating lymphocytes (TILs) from 2 unrelated patients, thus confirming its natural immunogenicity. We further investigated the neoantigen-specific clones and their T cell receptors (TCRs) via a combination of TCR sequencing, TCR overexpression, functional assays, and single-cell transcriptomics. Our analysis revealed a diverse repertoire of neoantigen-specific clones with both intra- and interpatient TCR similarities. Moreover, 1 dominant clone proved to cross-react with the highly prevalent RAS.Q61R variant. Transcriptome analysis revealed a high association of TCR clones with specific T cell phenotypes in response to cognate melanoma, with neoantigen-specific cells showing an activated and dysfunctional phenotype. Identification of recurrent neoantigens and their reactive TCRs can promote \"off-the-shelf\" precision immunotherapies, alleviating limitations of personalized treatments.

Spatially resolved transcriptomics was named Method of the Year 20201 and has continued to evolve rapidly since then, providing novel insights in development, physiology, and disease processes. Many approaches now offer excellent performance in formalin-fixed paraffin embedded tissue, opening up a wealth of archived clinical samples to these high-throughput spatial biology modalities. In this Chapter, we introduce a methodology to apply 10x Xenium In Situ to FFPE tissue microarrays. Using this method we have achieved a cost-effective approach to profile hundreds of genes with subcellular resolution in over a hundred human liver samples. Our method can easily be adapted to other tumour types, diseases, or model systems.