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Understanding tumor heterogeneity is a major challenge that was recognized as one of the first Cancer Grand Challenges, with a call to provide solutions to visualize tumor heterogeneity. The Rosetta team took on this challenge, exploiting advances in spatial-omics approaches centered around mass spectrometry imaging to map tumor heterogeneity at the cellular and molecular scales with different levels of resolution. See related article by Bressan et al., p. 16 See related article by Stratton et al., p. 22 See related article by Bhattacharjee et al., p. 28., (©2025 American Association for Cancer Research.)

From their early genesis, tumour cells integrate with the surrounding normal cells to form an abnormal structure that is tightly integrated with the host organism via blood and lymphatic vessels and even neural associations. Using these connections, emerging cancers send a plethora of mediators that efficiently perturb the entire organism and induce changes in distant tissues. These perturbations serendipitously favour early metastatic establishment by promoting a more favourable tissue environment (niche) that supports the persistence of disseminated tumour cells within a foreign tissue. Because the establishment of early metastatic niches represents a key limiting step for metastasis, the creation of a more suitable pre-conditioned tissue strongly enhances metastatic success. In this Review, we provide an updated view of the mechanisms and mediators of primary tumours described so far that induce a pro-metastatic conditioning of distant organs, which favours early metastatic niche formation. We reflect on the nature of cancer-induced systemic conditioning, considering that non-cancer-dependent perturbations of tissue homeostasis are also able to trigger pro-metastatic conditioning. We argue that a more holistic view of the processes catalysing metastatic progression is needed to identify preventive or therapeutic opportunities., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. Springer Nature Limited.)

Extrachromosomal DNA (ecDNA) is a major contributor to treatment resistance and poor outcome for patients with cancer 1,2 . Here we examine the diversity of ecDNA elements across cancer, revealing the associated tissue, genetic and mutational contexts. By analysing data from 14,778 patients with 39 tumour types from the 100,000 Genomes Project, we demonstrate that 17.1% of tumour samples contain ecDNA. We reveal a pattern highly indicative of tissue-context-based selection for ecDNAs, linking their genomic content to their tissue of origin. We show that not only is ecDNA a mechanism for amplification of driver oncogenes, but it also a mechanism that frequently amplifies immunomodulatory and inflammatory genes, such as those that modulate lymphocyte-mediated immunity and immune effector processes. Moreover, ecDNAs carrying immunomodulatory genes are associated with reduced tumour T cell infiltration. We identify ecDNAs bearing only enhancers, promoters and lncRNA elements, suggesting the combinatorial power of interactions between ecDNAs in trans. We also identify intrinsic and environmental mutational processes linked to ecDNA, including those linked to its formation, such as tobacco exposure, and progression, such as homologous recombination repair deficiency. Clinically, ecDNA detection was associated with tumour stage, more prevalent after targeted therapy and cytotoxic treatments, and associated with metastases and shorter overall survival. These results shed light on why ecDNA is a substantial clinical problem that can cooperatively drive tumour growth signals, alter transcriptional landscapes and suppress the immune system., (© 2024. The Author(s).)

Tumor-infiltrating B cells play a significant role in tumor development, progression, and prognosis, yet a comprehensive classification system is lacking. To address this gap, we present a pan-cancer single-cell RNA sequencing (scRNA-seq) atlas of tumor-infiltrating B and plasma cells across a large sample cohort. We identify key B cell subset signatures, revealing distinct subpopulations and highlighting the heterogeneity and functional diversity of these cells in the tumor microenvironment. We explore associations between B cell subsets and checkpoint inhibitor therapy responses, finding subset-specific effects on overall response. Additionally, we examine B and T cell crosstalk, identifying unique ligand-receptor pairs for specific B cell subsets, spatially validated. This comprehensive dataset serves as a valuable resource, providing a detailed atlas that enhances the understanding of B cell complexity in tumors and opens new avenues for research and therapeutic strategies., Competing Interests: Declaration of interests K.L. has the following disclosures (unrelated to this work): patent on indel burden and CPI response pending, patent on ctDNA minimal residual disease calling methods; speaker fees from Roche Tissue Diagnostics and Ellipses Pharma; research funding from CRUK TDL/Ono/LifeArc alliance and Genesis Therapeutics; and consulting roles with Monopteros Therapeutics, Kynos Therapeutics, SAGA Diagnostics, and Tempus Labs, Inc. Also unrelated to this work, K.L. is currently employed by Isomorphic Labs. J.L.R. has the following disclosures (unrelated to this work): patents filed for cancer early detection and consulting role for Achilles Therapeutics plc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tumors frequently display high chromosomal instability and contain multiple copies of genomic regions. Here, we describe Gain Route Identification and Timing In Cancer (GRITIC), a generic method for timing genomic gains leading to complex copy number states, using single-sample bulk whole-genome sequencing data. By applying GRITIC to 6,091 tumors, we found that non-parsimonious evolution is frequent in the formation of complex copy number states in genome-doubled tumors. We measured chromosomal instability before and after genome duplication in human tumors and found that late genome doubling was followed by an increase in the rate of copy number gain. Copy number gains often accumulate as punctuated bursts, commonly after genome doubling. We infer that genome duplications typically affect the landscape of copy number losses, while only minimally impacting copy number gains. In summary, GRITIC is a novel copy number gain timing framework that permits the analysis of copy number evolution in chromosomally unstable tumors. Significance: Complex genomic gains are associated with whole-genome duplications, which are frequent across tumors, span a large fraction of their genomes, and are linked to poorer outcomes. GRITIC infers when these gains occur during tumor development, which will help to identify the genetic events that drive tumor evolution. See related commentary by Taylor, p. 1766., (©2024 The Authors; Published by the American Association for Cancer Research.)

The advent of immunological therapies has revolutionized the treatment of solid and haematological cancers over the last decade. Licensed therapies which activate the immune system to target cancer cells can be broadly divided into two classes. The first class are antibodies that inhibit immune checkpoint signalling, known as immune checkpoint inhibitors (ICIs). The second class are cell-based immune therapies including chimeric antigen receptor T lymphocyte (CAR-T) cell therapies, natural killer (NK) cell therapies, and tumour infiltrating lymphocyte (TIL) therapies. The clinical efficacy of all these treatments generally outweighs the risks, but there is a high rate of immune-related adverse events (irAEs), which are often unpredictable in timing with clinical sequalae ranging from mild (e.g. rash) to severe or even fatal (e.g. myocarditis, cytokine release syndrome) and reversible to permanent (e.g. endocrinopathies).The mechanisms underpinning irAE pathology vary across different irAE complications and syndromes, reflecting the broad clinical phenotypes observed and the variability of different individual immune responses, and are poorly understood overall. Immune-related cardiovascular toxicities have emerged, and our understanding has evolved from focussing initially on rare but fatal ICI-related myocarditis with cardiogenic shock to more common complications including less severe ICI-related myocarditis, pericarditis, arrhythmias, including conduction system disease and heart block, non-inflammatory heart failure, takotsubo syndrome and coronary artery disease. In this scientific statement on the cardiovascular toxicities of immune therapies for cancer, we summarize the pathophysiology, epidemiology, diagnosis, and management of ICI, CAR-T, NK, and TIL therapies. We also highlight gaps in the literature and where future research should focus., (© 2024 European Society of Cardiology.)

Cancer is a major cause of global mortality, both in affluent countries and increasingly in developing nations. Many patients with cancer experience reduced life expectancy and have metastatic disease at the time of death. However, the more precise causes of mortality and patient deterioration before death remain poorly understood. This scarcity of information, particularly the lack of mechanistic insights, presents a challenge for the development of novel treatment strategies to improve the quality of, and potentially extend, life for patients with late-stage cancer. In addition, earlier deployment of existing strategies to prolong quality of life is highly desirable. In this Roadmap, we review the proximal causes of mortality in patients with cancer and discuss current knowledge about the interconnections between mechanisms that contribute to mortality, before finally proposing new and improved avenues for data collection, research and the development of treatment strategies that may improve quality of life for patients., (© 2024. Springer Nature Limited.)

Environmental carcinogens increase cancer incidence via both mutagenic and non-mutagenic mechanisms. There are over 500 known or suspected carcinogens classified by the International Agency for Research on Cancer. Sequencing of both cancerous and histologically non-cancerous tissue has been instrumental in improving our understanding of how environmental carcinogens cause cancer. Understanding how and defining which environmental or lifestyle exposures drive cancer will support cancer prevention. Recent research is revisiting the mechanisms of early tumorigenesis, paving the way for an era of molecular cancer prevention. Significance: Recent data have improved our understanding of how carcinogens cause cancer, which may reveal novel opportunities for molecular cancer prevention., (©2024 American Association for Cancer Research.)

While there is a great clinical need to understand the biology of metastatic cancer in order to treat it more effectively, research is hampered by limited sample availability. Research autopsy programmes can crucially advance the field through synchronous, extensive, and high-volume sample collection. However, it remains an underused strategy in translational research. Via an extensive questionnaire, we collected information on the study design, enrolment strategy, study conduct, sample and data management, and challenges and opportunities of research autopsy programmes in oncology worldwide. Fourteen programmes participated in this study. Eight programmes operated 24 h/7 days, resulting in a lower median postmortem interval (time between death and start of the autopsy, 4 h) compared with those operating during working hours (9 h). Most programmes (n = 10) succeeded in collecting all samples within a median of 12 h after death. A large number of tumour sites were sampled during each autopsy (median 15.5 per patient). The median number of samples collected per patient was 58, including different processing methods for tumour samples but also non-tumour tissues and liquid biopsies. Unique biological insights derived from these samples included metastatic progression, treatment resistance, disease heterogeneity, tumour dormancy, interactions with the tumour micro-environment, and tumour representation in liquid biopsies. Tumour patient-derived xenograft (PDX) or organoid (PDO) models were additionally established, allowing for drug discovery and treatment sensitivity assays. Apart from the opportunities and achievements, we also present the challenges related with postmortem sample collections and strategies to overcome them, based on the shared experience of these 14 programmes. Through this work, we hope to increase the transparency of postmortem tissue donation, to encourage and aid the creation of new programmes, and to foster collaborations on these unique sample collections. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Metabolic changes contribute to cancer initiation and progression through effects on cancer cells, the tumor microenvironment and whole-body metabolism. Alterations in serine metabolism and the control of one-carbon cycles have emerged as critical for the development of many tumor types. In this Review, we focus on the mitochondrial folate cycle. We discuss recent evidence that, in addition to supporting nucleotide synthesis, mitochondrial folate metabolism also contributes to metastasis through support of antioxidant defense, mitochondrial protein synthesis and the overflow of excess formate. These observations offer potential therapeutic opportunities, including the modulation of formate metabolism through dietary interventions and the use of circulating folate cycle metabolites as biomarkers for cancer detection., (© 2024. Springer Nature America, Inc.)

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis , which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

C-type lectin receptors (CLRs) expressed by myeloid cells constitute a versatile family of receptors that play a key role in innate immune recognition. Myeloid CLRs exhibit a remarkable ability to recognize an extensive array of ligands, from carbohydrates and beyond, and encompass pattern-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and markers of altered self. These receptors, classified into distinct subgroups, play pivotal roles in immune recognition and modulation of immune responses. Their intricate signaling pathways orchestrate a spectrum of cellular responses, influencing processes such as phagocytosis, cytokine production, and antigen presentation. Beyond their contributions to host defense in viral, bacterial, fungal, and parasitic infections, myeloid CLRs have been implicated in non-infectious diseases such as cancer, allergies, and autoimmunity. A nuanced understanding of myeloid CLR interactions with endogenous and microbial triggers is starting to uncover the context-dependent nature of their roles in innate immunity, with implications for therapeutic intervention., Competing Interests: Declaration of interests C.R.e.S. is a founder of Adendra Therapeutics and owns stock options and/or is a paid consultant for Adendra Therapeutics, Bicara Therapeutics, Montis Biosciences, and Bicycle Therapeutics, all unrelated to this work. C.R.e.S. also has an additional appointment as Visiting Professor in the Faculty of Medicine at Imperial College London and holds honorary professorships at University College London and King’s College London. G.D.B. holds an honorary professorship at the University of Cape Town., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adaptation to chronic hypoxia occurs through changes in protein expression, which are controlled by hypoxia-inducible factor 1α (HIF1α) and are necessary for cancer cell survival. However, the mechanisms that enable cancer cells to adapt in early hypoxia, before the HIF1α-mediated transcription programme is fully established, remain poorly understood. Here we show in human breast cancer cells, that within 3 h of hypoxia exposure, glycolytic flux increases in a HIF1α-independent manner but is limited by NAD + availability. Glycolytic ATP maintenance and cell survival in early hypoxia rely on reserve lactate dehydrogenase A capacity as well as the activity of glutamate-oxoglutarate transaminase 1 (GOT1), an enzyme that fuels malate dehydrogenase 1 (MDH1)-derived NAD + . In addition, GOT1 maintains low α-ketoglutarate levels, thereby limiting prolyl hydroxylase activity to promote HIF1α stabilisation in early hypoxia and enable robust HIF1α target gene expression in later hypoxia. Our findings reveal that, in normoxia, multiple enzyme systems maintain cells in a primed state ready to support increased glycolysis and HIF1α stabilisation upon oxygen limitation, until other adaptive processes that require more time are fully established., (© 2024. The Author(s).)

The last 50 years have witnessed extraordinary developments in understanding mechanisms of carcinogenesis, synthesized as the hallmarks of cancer. Despite this logical framework, our understanding of the molecular basis of systemic manifestations and the underlying causes of cancer-related death remains incomplete. Looking forward, elucidating how tumors interact with distant organs and how multifaceted environmental and physiological parameters impinge on tumors and their hosts will be crucial for advances in preventing and more effectively treating human cancers. In this perspective, we discuss complexities of cancer as a systemic disease, including tumor initiation and promotion, tumor micro- and immune macro-environments, aging, metabolism and obesity, cancer cachexia, circadian rhythms, nervous system interactions, tumor-related thrombosis, and the microbiome. Model systems incorporating human genetic variation will be essential to decipher the mechanistic basis of these phenomena and unravel gene-environment interactions, providing a modern synthesis of molecular oncology that is primed to prevent cancers and improve patient quality of life and cancer outcomes., Competing Interests: Declaration of interests C.A. reports employment at Saga Diagnostics and reports options to own stock in the company. C.A. and C.S. are listed as inventors on a European patent application relating to assay technology to detect tumor recurrence (PCT/GB2017/053289). This patent has been licensed to commercial entities and, under their terms of employment, C.A. and C.S. are due a revenue share of any revenue generated from such license(s). C.A. and C.S. declare a patent application (PCT/US2017/028013) for methods to detect lung cancer. C.A. and C.S. are named inventors on a patent application to determine methods and systems for tumor monitoring (PCT/EP2022/077987). C.A. and C.S. are named inventors on a provisional patent protection related to a ctDNA detection algorithm. J.A.J. has received honoraria for speaking at research symposia organized by Bristol Meyers Squibb and Glenmark Pharmaceuticals and previously served on the SAB of Pionyr Immunotherapeutics. D.H. is a founder and member of the BoD and SAB of Opna Bio, and a member of the SABs of Pfizer, Cellestia Biotech, 4D Molecular Therapeutics, and AtG Therapeutics. F.A. reports travel/accommodation/expenses from AstraZeneca, GlaxoSmithKline, Novartis, Pfizer, and Roche, and his institution has received research funding from AstraZeneca, Daiichi Sankyo, Lilly, Novartis, Pfizer, and Roche. C.M. Consultant/Advisory fees from Amgen, Astellas, AstraZeneca, Bayer, BeiGene, BMS, Celgene, Debiopharm, Genentech, Ipsen, Janssen, Lilly, MedImmune, MSD, Novartis, Pfizer, Roche, Sanofi, Orion. Principal/sub-Investigator of Clinical Trials for AbbVie, Aduro, Agios, Amgen, Argen-x, Astex, AstraZeneca, Aveo pharmaceuticals, Bayer, Beigene, Blueprint, BMS, Boeringer Ingelheim, Celgene, Chugai, Clovis, Daiichi Sankyo, Debiopharm, Eisai, Eos, Exelixis, Forma, Gamamabs, Genentech, Gortec, GSK, H3 biomedecine, Incyte, InnatePharma, Janssen, Kura Oncology, Kyowa, Lilly, Loxo, Lysarc, Lytix Biopharma, Medimmune, Menarini, Merus, MSD, Nanobiotix, Nektar Therapeutics, Novartis, Octimet, Oncoethix, Oncopeptides AB, Orion, Pfizer, Pharmamar, Pierre Fabre, Roche, Sanofi, Servier, Sierra Oncology, Taiho, Takeda, Tesaro, and Xencor. R.B. is founder of Oncosence. M.A.F. is founder and shareholder of Celesta Therapeutics. A.H. is a paid consultant for Calida Therapeutics. M.J.-H. has consulted for, and is a member of, the Achilles Therapeutics Scientific Advisory Board and Steering Committee, has received speaker honoraria from Pfizer, Astex Pharmaceuticals, Oslo Cancer Cluster, Bristol Myers Squibb, and is listed as a co-inventor on a European patent application relating to methods to detect lung cancer PCT/US2017/028013), this patent has been licensed to commercial entities and, under terms of employment, M.J.-H. is due a share of any revenue generated from such license(s). J.W.L. advises Restoration Foodworks, Nanocare Technologies, and Cornerstone Pharmaceuticals. S.L. received research funding to institution from Novartis, Bristol Myers Squibb, MSD, Puma Biotechnology, Eli Lilly, Nektar Therapeutics, AstraZeneca/Daiichi Sankyo, and Seattle Genetics. S.L. has acted as consultant (not compensated) to Seattle Genetics, Novartis, Bristol Myers Squibb, MSD, AstraZeneca/Daiichi Sankyo, Eli Lilly, Pfizer, Gilead Therapeutics, Nektar Therapeutics, PUMA Biotechnologies and Roche-Genentech. S.L. has acted as consultant (paid to institution) to Novartis, GlaxoSmithKline, Roche-Genentech, AstraZeneca/Daiichi Sankyo, Pfizer, Gilead Therapeutics, Seattle Genetics, MSD, Tallac Therapeutics, Eli Lilly, and Bristol Myers Squibb. I.M. is a consultant of LIfT BioSciences. K.M. has received honoraria from LEO Pharma, Pfizer and Bayer PLC. She has received research funding from LEO Pharma and Bayer PLC. S.Q. is a founder, CSO, and holds stock options in Achilles Therapeutics. C.S. acknowledges grants from AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Pfizer, Roche-Ventana, Invitae (previously Archer Dx Inc—collaboration in minimal residual disease sequencing technologies), Ono Pharmaceutical, and Personalis. He is Chief Investigator for the AZ MeRmaiD 1 and 2 clinical trials and is the Steering Committee Chair. He is also Co-Chief Investigator of the NHS Galleri trial funded by GRAIL and a paid member of GRAIL’s Scientific Advisory Board. He receives consultant fees from Achilles Therapeutics (also SAB member), Bicycle Therapeutics (also a SAB member), Genentech, Medicxi, China Innovation Centre of Roche (CICoR) formerly Roche Innovation Centre—Shanghai, Metabomed (until July 2022), Relay Therapeutics, and the Sarah Cannon Research Institute. C.S. has received honoraria from Amgen, AstraZeneca, Bristol Myers Squibb, GlaxoSmithKline, Illumina, MSD, Novartis, Pfizer, and Roche-Ventana. C.S. has previously held stock options in Apogen Biotechnologies and GRAIL, and currently has stock options in Epic Bioscience, Bicycle Therapeutics, and has stock options and is co-founder of Achilles Therapeutics. Patents: C.S. declares a patent application (PCT/US2017/028013) for methods to lung cancer); targeting neoantigens (PCT/EP2016/059401); identifying patent response to immune checkpoint blockade (PCT/EP2016/071471), determining HLA LOH (PCT/GB2018/052004); predicting survival rates of patients with cancer (PCT/GB2020/050221); identifying patients who respond to cancer treatment (PCT/GB2018/051912); methods for lung cancer detection (US20190106751A1). C.S. is an inventor on a European patent application (PCT/GB2017/053289) relating to assay technology to detect tumor recurrence. This patent has been licensed to a commercial entity and under their terms of employment C.S. is due a revenue share of any revenue generated from such license(s). K.H.V. is on the board of directors and shareholder of Bristol Myers Squibb and on the science advisory board (with stock options) of PMV Pharma, RAZE Therapeutics, Volastra Pharmaceuticals and Kovina Therapeutics. She is on the SAB of Ludwig Cancer and a co-founder and consultant of Faeth Therapeutics. She has been in receipt of research funding from Astex Pharmaceuticals and AstraZeneca and contributed to CRUK Cancer Research Technology filing of patent application WO/2017/144877. J.W. is an inventor on a US patent application (PCT/US17/53.717) submitted by the University of Texas MD Anderson Cancer Center which covers methods to enhance immune checkpoint blockade responses by modulating the microbiome, reports compensation for speaker’s bureau and honoraria from Imedex, Dava Oncology, Omniprex, Illumina, Gilead, PeerView, Physician Education Resource, MedImmune, Exelixis and Bristol Myers Squibb, and has served as a consultant/advisory board member for Roche/Genentech, Novartis, AstraZeneca, GlaxoSmithKline, Bristol Myers Squibb, Micronoma, OSE therapeutics, Merck, and Everimmune. Dr. Wargo receives stock options from Micronoma and OSE therapeutics. A.W. is on the board of Regain Therapeutics., (Copyright © 2024. Published by Elsevier Inc.)

The integration of cancer biomarkers into oncology has revolutionized cancer treatment, yielding remarkable advancements in cancer therapeutics and the prognosis of cancer patients. The development of personalized medicine represents a turning point and a new paradigm in cancer management, as biomarkers enable oncologists to tailor treatments based on the unique molecular profile of each patient's tumor. In this review, we discuss the scientific milestones of cancer biomarkers and explore future possibilities to improve the management of patients with solid tumors. This progress is primarily attributed to the biological characterization of cancers, advancements in testing methodologies, elucidation of the immune microenvironment, and the ability to profile circulating tumor fractions. Integrating these insights promises to continually advance the precision oncology field, fostering better patient outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mutational processes that alter large genomic regions occur frequently in developing tumors. They range from simple copy number gains and losses to the shattering and reassembly of entire chromosomes. These catastrophic events, such as chromothripsis, chromoplexy and the formation of extrachromosomal DNA, affect the expression of many genes and therefore have a substantial effect on the fitness of the cells in which they arise. In this review, we cover large genomic alterations, the mechanisms that cause them and their effect on tumor development and evolution., (© 2024. Springer Nature America, Inc.)

Cancer cells adapt and survive through the acquisition and selection of molecular modifications. This process defines cancer evolution. Building on a theoretical framework based on heritable genetic changes has provided insights into the mechanisms supporting cancer evolution. However, cancer hallmarks also emerge via heritable nongenetic mechanisms, including epigenetic and chromatin topological changes, and interactions between tumor cells and the tumor microenvironment. Recent findings on tumor evolutionary mechanisms draw a multifaceted picture where heterogeneous forces interact and influence each other while shaping tumor progression. A comprehensive characterization of the cancer evolutionary toolkit is required to improve personalized medicine and biomarker discovery., Significance: Tumor evolution is fueled by multiple enabling mechanisms. Importantly, genetic instability, epigenetic reprogramming, and interactions with the tumor microenvironment are neither alternative nor independent evolutionary mechanisms. As demonstrated by findings highlighted in this perspective, experimental and theoretical approaches must account for multiple evolutionary mechanisms and their interactions to ultimately understand, predict, and steer tumor evolution., (©2023 The Authors; Published by the American Association for Cancer Research.)

Intratumor heterogeneity provides the fuel for the evolution and selection of subclonal tumor cell populations. However, accurate inference of tumor subclonal architecture and reconstruction of tumor evolutionary histories from bulk DNA sequencing data remains challenging. Frequently, sequencing and alignment artifacts are not fully filtered out from cancer somatic mutations, and errors in the identification of copy number alterations or complex evolutionary events (e.g., mutation losses) affect the estimated cellular prevalence of mutations. Together, such errors propagate into the analysis of mutation clustering and phylogenetic reconstruction. In this Protocol, we present a new computational framework, CONIPHER (COrrecting Noise In PHylogenetic Evaluation and Reconstruction), that accurately infers subclonal structure and phylogenetic relationships from multisample tumor sequencing, accounting for both copy number alterations and mutation errors. CONIPHER has been used to reconstruct subclonal architecture and tumor phylogeny from multisample tumors with high-depth whole-exome sequencing from the TRACERx421 dataset, as well as matched primary-metastatic cases. CONIPHER outperforms similar methods on simulated datasets, and in particular scales to a large number of tumor samples and clones, while completing in under 1.5 h on average. CONIPHER enables automated phylogenetic analysis that can be effectively applied to large sequencing datasets generated with different technologies. CONIPHER can be run with a basic knowledge of bioinformatics and R and bash scripting languages., (© 2023. Springer Nature Limited.)

Greater collaboration needed to realize potential of molecular profiling initiatives for pediatric cancers.

Cells secrete extracellular vesicles (EVs) and non-vesicular extracellular (nano)particles (NVEPs or ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed to induce immune priming of antigen presenting cells or to be immuno-suppressive agents. We suspect that such disparate functions are due to variable compositions in EV subtypes and ENPs. We aimed to characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by many tumor cells. We established a protocol to separate small EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells. ENPs were poorly captured and did not affect dendritic cells. Small EVs specifically induced dendritic cell death. A mixed large/dense EV/VLP preparation was most efficient to induce dendritic cell maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by murine tumors and their contribution to tumor progression., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The transforming growth factor-β (TGFβ) family are a large group of evolutionarily conserved cytokines whose signalling modulates cell fate decision-making across varying cellular contexts at different stages of life. Here we discuss new findings in early embryos that reveal how, in contrast to our original understanding of morphogen interpretation, robust cell fate specification can originate from a noisy combination of signalling inputs and a broad range of signalling levels. We compare this evidence with novel findings on the roles of TGFβ family signalling in tissue maintenance and homeostasis during juvenile and adult life, spanning the skeletal, haemopoietic and immune systems. From these comparisons, it emerges that in contrast to robust developing systems, relatively small perturbations in TGFβ family signalling have detrimental effects at later stages in life, leading to aberrant cell fate specification and disease, for example in cancer or congenital disorders. Finally, we highlight novel strategies to target and amend dysfunction in signalling and discuss how gleaning knowledge from different fields of biology can help in the development of therapeutics for aberrant TGFβ family signalling in disease., (© 2023. Springer Nature Limited.)

Drugs that selectively kill senescent cells (senolytics) improve the outcomes of cancer, fibrosis and age-related diseases. Despite their potential, our knowledge of the molecular pathways that affect the survival of senescent cells is limited. To discover senolytic targets, we performed RNAi screens and identified coatomer complex I (COPI) vesicle formation as a liability of senescent cells. Genetic or pharmacological inhibition of COPI results in Golgi dispersal, dysfunctional autophagy, and unfolded protein response-dependent apoptosis of senescent cells, and knockdown of COPI subunits improves the outcomes of cancer and fibrosis in mouse models. Drugs targeting COPI have poor pharmacological properties, but we find that N-myristoyltransferase inhibitors (NMTi) phenocopy COPI inhibition and are potent senolytics. NMTi selectively eliminated senescent cells and improved outcomes in models of cancer and non-alcoholic steatohepatitis. Our results suggest that senescent cells rely on a hyperactive secretory apparatus and that inhibiting trafficking kills senescent cells with the potential to treat various senescence-associated diseases., (© 2023. The Author(s).)

Transmissible cancers are malignant cell lineages that spread clonally between individuals. Several such cancers, termed bivalve transmissible neoplasia (BTN), induce leukemia-like disease in marine bivalves. This is the case of BTN lineages affecting the common cockle, Cerastoderma edule, which inhabits the Atlantic coasts of Europe and northwest Africa. To investigate the evolution of cockle BTN, we collected 6,854 cockles, diagnosed 390 BTN tumors, generated a reference genome and assessed genomic variation across 61 tumors. Our analyses confirmed the existence of two BTN lineages with hemocytic origins. Mitochondrial variation revealed mitochondrial capture and host co-infection events. Mutational analyses identified lineage-specific signatures, one of which likely reflects DNA alkylation. Cytogenetic and copy number analyses uncovered pervasive genomic instability, with whole-genome duplication, oncogene amplification and alkylation-repair suppression as likely drivers. Satellite DNA distributions suggested ancient clonal origins. Our study illuminates long-term cancer evolution under the sea and reveals tolerance of extreme instability in neoplastic genomes., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Most computational methods that infer somatic copy number alterations (SCNAs) from bulk sequencing of DNA analyse tumour samples individually. However, the sequencing of multiple tumour samples from a patient's disease is an increasingly common practice. We introduce Refphase, an algorithm that leverages this multi-sampling approach to infer haplotype-specific copy numbers through multi-sample phasing. We demonstrate Refphase's ability to infer haplotype-specific SCNAs and characterise their intra-tumour heterogeneity, to uncover previously undetected allelic imbalance in low purity samples, and to identify parallel evolution in the context of whole genome doubling in a pan-cancer cohort of 336 samples from 99 tumours., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: CS acknowledges grant support from AstraZeneca, Boehringer-Ingelheim, BMS, Pfizer, Roche-Ventana, Invitae (previously Archer Dx, collaboration in minimal residual disease sequencing technologies), Ono Pharmaceutical, and Personalis; is an AstraZeneca advisory board member and chief investigator for the AZ MeRmaiD 1 and 2 clinical trials and is also co-chief investigator of the NHS Galleri trial funded by GRAIL and a paid member of GRAIL’s scientific advisory board; receives consultant fees from Achilles Therapeutics (also a scientific advisory board member), Bicycle Therapeutics (also a scientific advisory board member), Genentech, Medicxi, China Innovation Centre of Roche (CICoR) formerly Roche Innovation Centre – Shanghai, Metabomed (until July 2022) and the Sarah Cannon Research Institute; has received honoraria from Amgen, AstraZeneca, Pfizer, Novartis, GlaxoSmithKline, MSD, Bristol Myers Squibb, Illumina, and Roche-Ventana; had stock options in Apogen Biotechnologies and GRAIL until June 2021, and currently has stock options in Epic Bioscience, Bicycle Therapeutics, and has stock options and is co-founder of Achilles Therapeutics; is listed as an inventor on a European patent application relating to assay technology to detect tumour recurrence (PCT/GB2017/053289), the patent has been licensed to commercial entities and, under his terms of employment, CS is due a revenue share of any revenue generated from such license(s); holds patents relating to targeting neoantigens (PCT/EP2016/059401), identifying patient response to immune checkpoint blockade (PCT/EP2016/071471), determining HLA LOH (PCT/GB2018/052004), predicting survival rates of patients with cancer (PCT/GB2020/050221), identifying patients who respond to cancer treatment (PCT/GB2018/051912), a US patent relating to detecting tumour mutations (PCT/US2017/28013), methods for lung cancer detection (US20190106751A1) and both a European and US patent related to identifying insertion/deletion mutation targets (PCT/GB2018/051892) and is listed as a co-inventor on a patent application to determine methods and systems for tumour monitoring (PCT/EP2022/077987) and is a named inventor on a provisional patent protection related to a ctDNA detection algorithm. NM has received consultancy fees and has stock options in Achilles Therapeutics. NM holds European patents relating to targeting neoantigens (PCT/EP2016/ 059401), identifying patient response to immune checkpoint blockade (PCT/ EP2016/071471), determining HLA LOH (PCT/GB2018/052004), predicting survival rates of patients with cancer (PCT/GB2020/050221). All other authors declare no competing interests., (Copyright: © 2023 Watkins et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Immune evasion is a hallmark of cancer, enabling tumors to survive contact with the host immune system and evade the cycle of immune recognition and destruction. Here, we review the current understanding of the cancer cell-intrinsic factors driving immune evasion. We focus on T cells as key effectors of anti-cancer immunity and argue that cancer cells evade immune destruction by gaining control over pathways that usually serve to maintain physiological tolerance to self. Using this framework, we place recent mechanistic advances in the understanding of cancer immune evasion into broad categories of control over T cell localization, antigen recognition, and acquisition of optimal effector function. We discuss the redundancy in the pathways involved and identify knowledge gaps that must be overcome to better target immune evasion, including the need for better, routinely available tools that incorporate the growing understanding of evasion mechanisms to stratify patients for therapy and trials., Competing Interests: Declaration of interests E.G. has received consultant fees from Achilles Therapeutics. S.A.Q. is funded by a Cancer Research UK (CRUK) Senior Cancer Research Fellowship (C36463/A22246) and a CRUK Biotherapeutic Program grant (C36463/A20764) and receives support from the CRUK-UCL Centre (C416/A18088), Cancer Research UK City of London Centre Award (C7893/A26233), and the Cancer Immuno-therapy Accelerator Award (CITA-CRUK) (C33499/A20265). C.S. acknowledges grant support from AstraZeneca, Boehringer-Ingelheim, Bristol Myers Squibb, Pfizer, Roche-Ventana, Invitae (previously Archer Dx Inc.—collaboration in minimal residual disease sequencing technologies), Ono Pharmaceutical, and Personalis. He is an AstraZeneca Advisory Board member and Chief Investigator for the AZ MeRmaiD 1 and 2 clinical trials and is also co-chief investigator of the NHS Galleri trial funded by GRAIL and a paid member of GRAIL’s Scientific Advisory Board. He receives consultant fees from Achilles Therapeutics (also a SAB member), Bicycle Therapeutics (also a SAB member), Genentech, Medicxi, China Innovation Center of Roche (CICoR), formerly Roche Innovation Center, Shanghai, Metabomed (until July 2022), and the Sarah Cannon Research Institute. C.S. has received honoraria from Amgen, AstraZeneca, Pfizer, Novartis, GlaxoSmithKline, MSD, Bristol Myers Squibb, Illumina, and Roche-Ventana. C.S. had stock options in Apogen Biotechnologies and GRAIL until June 2021 and currently has stock options in Epic Bioscience, Bicycle Therapeutics, and has stock options and is co-founder of Achilles Therapeutics. C.S. is an inventor on a European patent application relating to assay technology to detect tumor recurrence (PCT/GB2017/053289); the patent has been licensed to commercial entities and under his terms of employment. C.S. is due a revenue share of any revenue generated from such license(s). C.S. holds patents relating to targeting neoantigens (PCT/EP2016/059401), identifying patient response to immune checkpoint blockade (PCT/EP2016/071471), determining HLA LOH (PCT/GB2018/052004), predicting survival rates of patients with cancer (PCT/GB2020/050221), identifying patients who respond to cancer treatment (PCT/GB2018/051912), a US patent relating to detecting tumor mutations (PCT/US2017/28013), methods for lung cancer detection (US20190106751A1), and both a European and US patent related to identifying insertion/deletion mutation targets (PCT/GB2018/051892) and is co-inventor to a patent application to determine methods and systems for tumor monitoring (PCT/EP2022/077987). S.A.Q. is co-founder, C.S.O., and holds stock options in Achilles Therapeutics. S.A.Q. is also an inventor in patents describing the use of anti-CD25 antibodies to target Tregs and receives milestones related to these patents., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Previous studies have revealed a role for proline metabolism in supporting cancer development and metastasis. In this study, we show that many cancer cells respond to loss of attachment by accumulating and secreting proline. Detached cells display reduced proliferation accompanied by a general decrease in overall protein production and de novo amino acid synthesis compared to attached cells. However, proline synthesis was maintained under detached conditions. Furthermore, while overall proline incorporation into proteins was lower in detached cells compared to other amino acids, there was an increased production of the proline-rich protein collagen. The increased excretion of proline from detached cells was also shown to be used by macrophages, an abundant and important component of the tumor microenvironment. Our study suggests that detachment induced accumulation and secretion of proline may contribute to tumor progression by supporting increased production of extracellular matrix and providing proline to surrounding stromal cells.

Immune checkpoint inhibitors (ICIs) have fundamentally changed the treatment landscape of various cancers. While ICI treatments result in improved survival, quality of life and are cost-effective, the majority of patients experience at least one immune-related adverse event (irAE). Many of these side effects cause little discomfort or are asymptomatic; however, irAEs can affect any organ and are potentially life-threatening. Consequently, early diagnosis and appropriate treatment of irAEs are critical for optimizing long-term outcomes and quality of life in affected patients. Some irAEs are diagnosed according to typical symptoms, others by abnormal findings from diagnostic tests. While there are various guidelines addressing the management of irAEs, recommendations for the early recognition of irAEs as well as the optimal extent and frequency of laboratory tests are mostly lacking. In clinical practice, blood sampling is usually performed before each ICI administration (i.e., every 2-3 weeks), often for several months, representing a burden for patients as well as health care systems. In this report, we propose essential laboratory and functional tests to improve the early detection and management of irAEs and in cancer patients treated with ICIs. These multidisciplinary expert recommendations regarding essential laboratory and functional tests can be used to identify possible irAEs at an early time point, initiate appropriate interventions to improve patient outcomes, and reduce the burden of blood sampling during ICI treatment., (© 2023. The Author(s).)

Tumor-infiltrating T cells offer a promising avenue for cancer treatment, yet their states remain to be fully characterized. Here we present a single-cell atlas of T cells from 308,048 transcriptomes across 16 cancer types, uncovering previously undescribed T cell states and heterogeneous subpopulations of follicular helper, regulatory and proliferative T cells. We identified a unique stress response state, T STR , characterized by heat shock gene expression. T STR cells are detectable in situ in the tumor microenvironment across various cancer types, mostly within lymphocyte aggregates or potential tertiary lymphoid structures in tumor beds or surrounding tumor edges. T cell states/compositions correlated with genomic, pathological and clinical features in 375 patients from 23 cohorts, including 171 patients who received immune checkpoint blockade therapy. We also found significantly upregulated heat shock gene expression in intratumoral CD4/CD8 + cells following immune checkpoint blockade treatment, particularly in nonresponsive tumors, suggesting a potential role of T STR cells in immunotherapy resistance. Our well-annotated T cell reference maps, web portal and automatic alignment/annotation tool could provide valuable resources for T cell therapy optimization and biomarker discovery., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Metastasis is a complex process and the leading cause of cancer-related death globally. Recent studies have demonstrated that genomic sequencing data from paired primary and metastatic tumours can be used to trace the evolutionary origins of cells responsible for metastasis. This approach has yielded new insights into the genomic alterations that engender metastatic potential, and the mechanisms by which cancer spreads. Given that the reliability of these approaches is contingent upon how representative the samples are of primary and metastatic tumour heterogeneity, we review insights from studies that have reconstructed the evolution of metastasis within the context of their cohorts and designs. We discuss the role of research autopsies in achieving the comprehensive sampling necessary to advance the current understanding of metastasis., Competing Interests: Declaration of interests S.H., P.F., and S.Z. have no conflicts of interests to declare. M.J-H. has consulted for, and is a member of, the Achilles Therapeutics Scientific Advisory Board and Steering Committee, has received speaker honoraria from Pfizer, Astex Pharmaceuticals, Oslo Cancer Cluster, and holds a patent PCT/US2017/028013 relating to methods for lung cancer detection. C.S. is an AstraZeneca Advisory Board Member and Chief Investigator for the MeRmaiD1 clinical trial and has consulted for Amgen, Pfizer, Novartis, GlaxoSmithKline, MSD, Bristol Myers Squibb, AstraZeneca, Illumina, Genentech, Roche-Ventana, GRAIL, Medicxi, Bicycle Therapeutics, Metabomed, and the Sarah Cannon Research Institute; has stock options in ApoGen Biotechnologies, Epic Bioscience, and GRAIL; and has stock options and is co-founder of Achilles Therapeutics. C.S. holds patents relating to assay technology to detect tumour recurrence (PCT/GB2017/053289); to targeting neoantigens (PCT/EP2016/059401), identifying patent response to immune checkpoint blockade (PCT/EP2016/071471), determining HLA LOH (PCT/GB2018/052004), predicting survival rates of patients with cancer (PCT/GB2020/050221), to treating cancer by targeting insertion/deletion mutations (PCT/GB2018/051893), identifying insertion/deletion mutation targets (PCT/GB2018/051892); methods for lung cancer detection (PCT/US2017/028013), identifying responders to cancer treatment (PCT/GB2018/051912); and a patent application to determine methods and systems for tumour monitoring (PCT/EP2022/077987)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell migration is crucial for cancer dissemination. We find that AMP-activated protein kinase (AMPK) controls cell migration by acting as an adhesion sensing molecular hub. In 3-dimensional matrices, fast-migrating amoeboid cancer cells exert low adhesion/low traction linked to low ATP/AMP, leading to AMPK activation. In turn, AMPK plays a dual role controlling mitochondrial dynamics and cytoskeletal remodelling. High AMPK activity in low adhering migratory cells, induces mitochondrial fission, resulting in lower oxidative phosphorylation and lower mitochondrial ATP. Concurrently, AMPK inactivates Myosin Phosphatase, increasing Myosin II-dependent amoeboid migration. Reducing adhesion or mitochondrial fusion or activating AMPK induces efficient rounded-amoeboid migration. AMPK inhibition suppresses metastatic potential of amoeboid cancer cells in vivo, while a mitochondrial/AMPK-driven switch is observed in regions of human tumours where amoeboid cells are disseminating. We unveil how mitochondrial dynamics control cell migration and suggest that AMPK is a mechano-metabolic sensor linking energetics and the cytoskeleton., (© 2023. The Author(s).)

Purpose: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy., Experimental Design: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo., Results: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone., Conclusions: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy., (©2023 The Authors; Published by the American Association for Cancer Research.)

Recent identification of oncogenic cells within healthy tissues and the prevalence of indolent cancers found incidentally at autopsies reveal a greater complexity in tumor initiation than previously appreciated. The human body contains roughly 40 trillion cells of 200 different types that are organized within a complex three-dimensional matrix, necessitating exquisite mechanisms to restrain aberrant outgrowth of malignant cells that have the capacity to kill the host. Understanding how this defense is overcome to trigger tumorigenesis and why cancer is so extraordinarily rare at the cellular level is vital to future prevention therapies. In this review, we discuss how early initiated cells are protected from further tumorigenesis and the non-mutagenic pathways by which cancer risk factors promote tumor growth. By nature, the absence of permanent genomic alterations potentially renders these tumor-promoting mechanisms clinically targetable. Finally, we consider existing strategies for early cancer interception with perspectives on the next steps for molecular cancer prevention., Competing Interests: Declaration of interests C.S. acknowledges grant support from AstraZeneca, Boehringer-Ingelheim, Bristol Myers Squibb, Pfizer, Roche-Ventana, Invitae (previously Archer Dx Inc - collaboration in minimal residual disease sequencing technologies), Ono Pharmaceutical, and Personalis. He is an AstraZeneca Advisory Board member and Chief Investigator for the AZ MeRmaiD 1 and 2 clinical trials and is also Co-Chief Investigator of the NHS Galleri trial funded by GRAIL and a paid member of GRAIL’s Scientific Advisory Board. He receives consultant fees from Achilles Therapeutics (also a SAB member), Bicycle Therapeutics (also a SAB member), Genentech, Medicxi, China Innovation Center of Roche (CICoR) formerly Roche Innovation Center – Shanghai, Metabomed (until July 2022), and the Sarah Cannon Research Institute. C.S. has received honoraria from Amgen, AstraZeneca, Pfizer, Novartis, GlaxoSmithKline, MSD, Bristol Myers Squibb, Illumina, and Roche-Ventana. C.S. had stock options in Apogen Biotechnologies and GRAIL until June 2021, and currently has stock options in Epic Bioscience, Bicycle Therapeutics, and has stock options and is co-founder of Achilles Therapeutics. C.S. is an inventor on a European patent application relating to assay technology to detect tumor recurrence (PCT/GB2017/053289), the patent has been licensed to commercial entities, and under his terms of employment, C.S. is due a revenue share of any revenue generated from such license(s). C.S. holds patents relating to targeting neoantigens (PCT/EP2016/059401), identifying patient response to immune checkpoint blockade (PCT/EP2016/071471), determining HLA LOH (PCT/GB2018/052004), predicting survival rates of patients with cancer (PCT/GB2020/050221), identifying patients who respond to cancer treatment (PCT/GB2018/051912), a US patent relating to detecting tumor mutations (PCT/US2017/28013), methods for lung cancer detection (US20190106751A1) and both a European and US patent related to identifying insertion/deletion mutation targets (PCT/GB2018/051892), and is co-inventor to a patent application to determine methods and systems for tumor monitoring (PCT/EP2022/077987)., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Dissecting mechanisms driving subclone expansion in primary cancers has been challenging. Here, we present a protocol to systematically disrupt entire gene networks and assess the functional impact of this perturbation on cancer cell fitness. By combining arrayed CRISPR libraries and high-content microscopy, we describe steps to identify classes of genes whose inactivation promotes resistance to environmental challenges faced by cancer cells during tumor growth or upon therapy. A proof-of-principle interrogation of the epigenetic regulatory network is described. For complete details on the use and execution of this protocol, please refer to Loukas et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Conventional dendritic cells type 1 (cDC1) are critical for inducing protective CD8 + T cell responses to tumour and viral antigens. In many instances, cDC1 access those antigens in the form of material internalised from dying tumour or virally-infected cells. How cDC1 extract dead cell-associated antigens and cross-present them in the form of peptides bound to MHC class I molecules to CD8 + T cells remains unclear. Here we review the biology of dendritic cell natural killer group receptor-1 (DNGR-1; also known as CLEC9A), a C-type lectin receptor highly expressed on cDC1 that plays a key role in this process. We highlight recent advances that support a function for DNGR-1 signalling in promoting inducible rupture of phagocytic or endocytic compartments containing dead cell debris, thereby making dead cell-associated antigens accessible to the endogenous MHC class I processing and presentation machinery of cDC1. We further review how DNGR-1 detects dead cells, as well as the functions of the receptor in anti-viral and anti-tumour immunity. Finally, we highlight how the study of DNGR-1 has opened new perspectives into cross-presentation, some of which may have applications in immunotherapy of cancer and vaccination against viral diseases., Competing Interests: Declaration of Competing Interest C.R.S. is a founder of Adendra Therapeutics and owns stock options and/or is a paid consultant for Adendra Therapeutics, Bicara Therapeutics, Montis Biosciences, Bicycle Therapeutics and Sosei Heptares. C.R.S. has an additional appointment as a Visiting Professor in the Faculty of Medicine at Imperial College London and holds honorary professorships at University College London and King’s College London., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hematological malignancies are a highly heterogeneous group of diseases with varied molecular and phenotypical characteristics. SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complexes play significant roles in the regulation of gene expression, being essential for processes such as cell maintenance and differentiation in hematopoietic stem cells. Furthermore, alterations in SWI/SNF complex subunits, especially in ARID1A/1B/2, SMARCA2/4, and BCL7A, are highly recurrent across a wide variety of lymphoid and myeloid malignancies. Most genetic alterations cause a loss of function of the subunit, suggesting a tumor suppressor role. However, SWI/SNF subunits can also be required for tumor maintenance or even play an oncogenic role in certain disease contexts. The recurrent alterations of SWI/SNF subunits highlight not only the biological relevance of SWI/SNF complexes in hematological malignancies but also their clinical potential. In particular, increasing evidence has shown that mutations in SWI/SNF complex subunits confer resistance to several antineoplastic agents routinely used for the treatment of hematological malignancies. Furthermore, mutations in SWI/SNF subunits often create synthetic lethality relationships with other SWI/SNF or non-SWI/SNF proteins that could be exploited therapeutically. In conclusion, SWI/SNF complexes are recurrently altered in hematological malignancies and some SWI/SNF subunits may be essential for tumor maintenance. These alterations, as well as their synthetic lethal relationships with SWI/SNF and non-SWI/SNF proteins, may be pharmacologically exploited for the treatment of diverse hematological cancers., (© 2023. The Author(s).)

Whole-genome duplication (WGD) is a frequent event in cancer evolution and an important driver of aneuploidy. The role of the p53 tumor suppressor in WGD has been enigmatic: p53 can block the proliferation of tetraploid cells, acting as a barrier to WGD, but can also promote mitotic bypass, a key step in WGD via endoreduplication. In wild-type (WT) p53 tumors, WGD is frequently associated with activation of the E2F pathway, especially amplification of CCNE1, encoding cyclin E1. Here, we show that elevated cyclin E1 expression causes replicative stress, which activates ATR- and Chk1-dependent G2 phase arrest. p53, via its downstream target p21, together with Wee1, then inhibits mitotic cyclin-dependent kinase activity sufficiently to activate APC/C Cdh1 and promote mitotic bypass. Cyclin E expression suppresses p53-dependent senescence after mitotic bypass, allowing cells to complete endoreduplication. Our results indicate that p53 can contribute to cancer evolution through the promotion of WGD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Disparities in cancer screening and outcomes based on factors such as sex, socioeconomic status, and race and ethnicity in the United States are well documented. A blood-based multi-cancer early detection (MCED) test that detects a shared cancer signal across multiple cancer types and also predicts the cancer signal origin was developed and validated in the Circulating Cell-free Genome Atlas study (CCGA; NCT02889978). CCGA is a prospective, multicenter, case-control, observational study with longitudinal follow-up (overall N = 15,254). In this pre-specified, exploratory, descriptive analysis, test performance was evaluated among racial and ethnic groups. Overall, 4077 participants comprised the independent validation set with confirmed cancer status (cancer: n = 2823; non-cancer: n = 1254). Participants were stratified into the following racial/ethnic groups: Black (non-Hispanic), Hispanic (all races), Other (non-Hispanic), Other/unknown and White (non-Hispanic). Cancer and non-cancer participants were predominantly White (n = 2316, 82.0% and n = 996, 79.4%, respectively). Across groups, specificity for cancer signal detection ranged from 98.1% [n = 103; 95% CI: 93.2-99.5%] to 100% [n = 85; 95% CI: 95.7-100.0%]. The sensitivity for cancer signal detection across groups ranged from 43.9% [n = 57; 95% CI: 31.8-56.7%] to 63.0% [n = 192; 95% CI: 56.0-69.5%] and generally increased with clinical stage. The MCED test had consistently high specificity and similar sensitivity across racial and ethnic groups, though results are limited by sample size for some groups. Results support the broad applicability of this MCED test and clinical implementation on a population scale as a complement to standard screening., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The evolution of established cancers is driven by selection of cells with enhanced fitness. Subclonal mutations in numerous epigenetic regulator genes are common across cancer types, yet their functional impact has been unclear. Here, we show that disruption of the epigenetic regulatory network increases the tolerance of cancer cells to unfavorable environments experienced within growing tumors by promoting the emergence of stress-resistant subpopulations. Disruption of epigenetic control does not promote selection of genetically defined subclones or favor a phenotypic switch in response to environmental changes. Instead, it prevents cells from mounting an efficient stress response via modulation of global transcriptional activity. This "transcriptional numbness" lowers the probability of cell death at early stages, increasing the chance of long-term adaptation at the population level. Our findings provide a mechanistic explanation for the widespread selection of subclonal epigenetic-related mutations in cancer and uncover phenotypic inertia as a cellular trait that drives subclone expansion., Competing Interests: Declaration of interests H.P. is currently an employee of Seqera Labs. E.S. is a member of the advisory board of Phenomic, consults for Theolytics and receives funding from Merck Sharp & Dohme., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

POLQ is a key effector of DSB repair by microhomology-mediated end-joining (MMEJ) and is overexpressed in many cancers. POLQ inhibitors confer synthetic lethality in HR and Shieldin-deficient cancer cells, which has been proposed to reflect a critical dependence on the DSB repair pathway by MMEJ. Whether POLQ also operates independent of MMEJ remains unexplored. Here, we show that POLQ-deficient cells accumulate post-replicative ssDNA gaps upon BRCA1/2 loss or PARP inhibitor treatment. Biochemically, cooperation between POLQ helicase and polymerase activities promotes RPA displacement and ssDNA-gap fill-in, respectively. POLQ is also capable of microhomology-mediated gap skipping (MMGS), which generates deletions during gap repair that resemble the genomic scars prevalent in POLQ overexpressing cancers. Our findings implicate POLQ in mutagenic post-replicative gap sealing, which could drive genome evolution in cancer and whose loss places a critical dependency on HR for gap protection and repair and cellular viability., Competing Interests: Declaration of interests S.J.B is a co-founder, VP Science Strategy and a shareholder of Artios Pharma Ltd. G.S.H. received consultancy fees from and is a shareholder of Artios Pharma Ltd and is a member of the Molecular Cell advisory board. V.G., J.N., H.M.R.R., and G.C.M.S. are all employees and shareholders of Artios Pharma Ltd. G.C.M.S. is a shareholder of AstraZeneca PLC., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

A fundamental requirement for cancer initiation is the activation of developmental programmes by mutant cells. Oncogenic signals often confer an undifferentiated, stem cell-like phenotype that supports the long-term proliferative potential of cancer cells. Although cancer is a genetically driven disease, mutations in cancer-driver genes alone are insufficient for tumour formation, and the proliferation of cells harbouring oncogenic mutations depends on their microenvironment. In this Opinion article we discuss how the reprogrammed status of cancer cells not only represents the essence of their tumorigenicity but triggers 'reflected stemness' in their surrounding normal counterparts. We propose that this reciprocal interaction underpins the establishment of the tumour microenvironment (TME)., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

Background: Seemingly normal tissues progressively become populated by mutant clones over time. Most of these clones bear mutations in well-known cancer genes but only rarely do they transform into cancer. This poses questions on what triggers cancer initiation and what implications somatic variation has for cancer early detection., Design: We analyzed recent mutational screens of healthy and cancer-free diseased tissues to compare somatic drivers and the causes of somatic variation across tissues. We then reviewed the mechanisms of clonal expansion and their relationships with age and diseases other than cancer. We finally discussed the relevance of somatic variation for cancer initiation and how it can help or hinder cancer detection and prevention., Results: The extent of somatic variation is highly variable across tissues and depends on intrinsic features, such as tissue architecture and turnover, as well as the exposure to endogenous and exogenous insults. Most somatic mutations driving clonal expansion are tissue-specific and inactivate tumor suppressor genes involved in chromatin modification and cell growth signaling. Some of these genes are more frequently mutated in normal tissues than cancer, indicating a context-dependent cancer-promoting or -protective role. Mutant clones can persist over a long time or disappear rapidly, suggesting that their fitness depends on the dynamic equilibrium with the environment. The disruption of this equilibrium is likely responsible for their transformation into malignant clones and knowing what triggers this process is key for cancer prevention and early detection. Somatic variation should be considered in liquid biopsy, where it may contribute cancer-independent mutations, and in the identification of cancer drivers, since not all mutated genes favoring clonal expansion also drive tumorigenesis., Conclusion: Somatic variation and the factors governing homeostasis of normal tissues should be taken into account when devising strategies for cancer prevention and early detection., Competing Interests: Disclosure The authors have declared no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

This study offers longitudinal insight into the impact of three SARS-CoV-2 vaccinations on humoral and cellular immunity in patients with solid cancers, patients with hematologic malignancies, and persons without cancer. For all cohorts, virus-neutralizing immunity was significantly depleted over a period of up to 9 months following the second vaccine dose, the one striking exception being IL2 production by SARS-CoV-2 antigen-specific T cells. Immunity was restored by the third vaccine dose, except in a substantial number of patients with hematologic malignancy, for whom both cancer type and treatment schedule were associated with nonresponse. Thus, whereas most patients with myelodysplastic syndrome were conspicuously good responders, some patients with other hematologic malignancies receiving cancer therapies within 2 weeks of vaccination showed no seroconversion despite three vaccine doses. Moreover, SARS-CoV-2 exposure during the course of the study neither prevented immunity waning, even in healthy controls, nor guaranteed vaccine responsiveness. These data offer real-world human immunologic insights that can inform health policy for patients with cancer., Competing Interests: Authors’ Disclosures P. Patten reports grants from Gilead, Roche; personal fees from Janssen, AstraZeneca, Abbvie, Beigene, and Novartis outside the submitted work. A.C. Hayday is co-founder, board member, and equity holder in ImmunoQure AG. This company is interested in antibodies but has no infectious disease or vaccine programs so cannot objectively be in conflict of interest with the work pursued in the study submitted to you; co-founder, board member, and equity holder in Gamma Delta Therapeutics. This company which no longer exists, was interested in gamma delta T cell which are an immunologic components, but the company has no infectious disease or vaccine programs so cannot objectively be in conflict of interest with the work pursued in the study submitted to you; co-founder, board member, and equity holder in Adaptate Biotherapeutics. This company, which no longer exists, was interested in gamma delta T cells which are an immunologic components, but the company has no infectious disease or vaccine programs so cannot objectively be in conflict of interest with the work pursued in the study submitted to you. A.C. Hayday receives research funding from Marengo Inc but this is a T-cell company with no vaccine program. A.C. Hayday receives research funding from Takeda Inc but this is funding solely relates to the use of gamma delta T cells for cancer immunotherapy. No disclosures were reported by the other authors.

Activation of cellular hypoxia pathways, orchestrated by HIF (hypoxia-inducible factor) transcription factors, is a common feature of multiple tumor types, resulting from microenvironment factors and oncogenic mutation. Although they help drive many of the "hallmarks" of cancer and are associated with poor outcome and resistance to therapy, the transcriptional targets of HIF vary considerably depending on the cell type. By integrating 72 genome-wide assays of HIF binding and transcriptional regulation from multiple cancer types, we define a consensus set of 48 HIF target genes that is highly conserved across cancer types and cell lineages. These genes provide an effective marker of HIF activation in bulk and single-cell transcriptomic analyses across a wide range of cancer types and in malignant and stromal cell types. This allows the tissue-orchestrated responses to the hypoxic tumor microenvironment and to oncogenic HIF activation to be deconvoluted at the tumor and single-cell level., Competing Interests: Declaration of interests P.J.R. is a scientific co-founder of ReOx Ltd., a company developing inhibitors of the HIF hydroxylase enzymes., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Aneuploidy, chromosomal instability, somatic copy-number alterations, and whole-genome doubling (WGD) play key roles in cancer evolution and provide information for the complex task of phylogenetic inference. We present MEDICC2, a method for inferring evolutionary trees and WGD using haplotype-specific somatic copy-number alterations from single-cell or bulk data. MEDICC2 eschews simplifications such as the infinite sites assumption, allowing multiple mutations and parallel evolution, and does not treat adjacent loci as independent, allowing overlapping copy-number events. Using simulations and multiple data types from 2780 tumors, we use MEDICC2 to demonstrate accurate inference of phylogenies, clonal and subclonal WGD, and ancestral copy-number states., (© 2022. The Author(s).)

Glioblastoma evolution is facilitated by intratumour heterogeneity, which poses a major hurdle to effective treatment. Evidence indicates a key role for oncogene amplification on extrachromosomal DNA (ecDNA) in accelerating tumour evolution and thus resistance to treatment, particularly in glioblastomas. Oncogenes contained within ecDNA can reach high copy numbers and expression levels, and their unequal segregation can result in more rapid copy number changes in response to therapy than is possible through natural selection of intrachromosomal genomic loci. Notably, targeted therapies inhibiting oncogenic pathways have failed to improve glioblastoma outcomes. In this Perspective, we outline reasons for this disappointing lack of clinical translation and present the emerging evidence implicating ecDNA as an important driver of tumour evolution. Furthermore, we suggest that through detection of ecDNA, patient selection for clinical trials of novel agents can be optimized to include those most likely to benefit based on current understanding of resistance mechanisms. We discuss the challenges to successful translation of this approach, including accurate detection of ecDNA in tumour tissue with novel technologies, development of faithful preclinical models for predicting the efficacy of novel agents in the presence of ecDNA oncogenes, and understanding the mechanisms of ecDNA formation during cancer evolution and how they could be attenuated therapeutically. Finally, we evaluate the feasibility of routine ecDNA characterization in the clinic and how this process could be integrated with other methods of molecular stratification to maximize the potential for clinical translation of precision medicines., (© 2022. Springer Nature Limited.)

Extrachromosomal DNA (ecDNA) is a common mode of oncogene amplification but is challenging to analyze. Here, we adapt CRISPR-CATCH, in vitro CRISPR-Cas9 treatment and pulsed field gel electrophoresis of agarose-entrapped genomic DNA, previously developed for bacterial chromosome segments, to isolate megabase-sized human ecDNAs. We demonstrate strong enrichment of ecDNA molecules containing EGFR, FGFR2 and MYC from human cancer cells and NRAS ecDNA from human metastatic melanoma with acquired therapeutic resistance. Targeted enrichment of ecDNA versus chromosomal DNA enabled phasing of genetic variants, identified the presence of an EGFRvIII mutation exclusively on ecDNAs and supported an excision model of ecDNA genesis in a glioblastoma model. CRISPR-CATCH followed by nanopore sequencing enabled single-molecule ecDNA methylation profiling and revealed hypomethylation of the EGFR promoter on ecDNAs. We distinguished heterogeneous ecDNA species within the same sample by size and sequence with base-pair resolution and discovered functionally specialized ecDNAs that amplify select enhancers or oncogene-coding sequences., (© 2022. The Author(s).)

Single-cell RNA sequencing studies have suggested that total mRNA content correlates with tumor phenotypes. Technical and analytical challenges, however, have so far impeded at-scale pan-cancer examination of total mRNA content. Here we present a method to quantify tumor-specific total mRNA expression (TmS) from bulk sequencing data, taking into account tumor transcript proportion, purity and ploidy, which are estimated through transcriptomic/genomic deconvolution. We estimate and validate TmS in 6,590 patient tumors across 15 cancer types, identifying significant inter-tumor variability. Across cancers, high TmS is associated with increased risk of disease progression and death. TmS is influenced by cancer-specific patterns of gene alteration and intra-tumor genetic heterogeneity as well as by pan-cancer trends in metabolic dysregulation. Taken together, our results indicate that measuring cell-type-specific total mRNA expression in tumor cells predicts tumor phenotypes and clinical outcomes., (© 2022. The Author(s).)

Background: A major driver of cancer chromosomal instability is replication stress, the slowing or stalling of DNA replication. How replication stress and genomic instability are connected is not known. Aphidicolin-induced replication stress induces breakages at common fragile sites, but the exact causes of fragility are debated, and acute genomic consequences of replication stress are not fully explored., Results: We characterize DNA copy number alterations (CNAs) in single, diploid non-transformed cells, caused by one cell cycle in the presence of either aphidicolin or hydroxyurea. Multiple types of CNAs are generated, associated with different genomic regions and features, and observed copy number landscapes are distinct between aphidicolin and hydroxyurea-induced replication stress. Coupling cell type-specific analysis of CNAs to gene expression and single-cell replication timing analyses pinpointed the causative large genes of the most recurrent chromosome-scale CNAs in aphidicolin. These are clustered on chromosome 7 in RPE1 epithelial cells but chromosome 1 in BJ fibroblasts. Chromosome arm level CNAs also generate acentric lagging chromatin and micronuclei containing these chromosomes., Conclusions: Chromosomal instability driven by replication stress occurs via focal CNAs and chromosome arm scale changes, with the latter confined to a very small subset of chromosome regions, potentially heavily skewing cancer genome evolution. Different inducers of replication stress lead to distinctive CNA landscapes providing the opportunity to derive copy number signatures of specific replication stress mechanisms. Single-cell CNA analysis thus reveals the impact of replication stress on the genome, providing insights into the molecular mechanisms which fuel chromosomal instability in cancer., (© 2022. The Author(s).)