MHC Hammer reveals genetic and non-genetic HLA disruption in cancer evolution.

Disruption of the class I human leukocyte antigen (HLA) molecules has important implications for immune evasion and tumor evolution. We developed major histocompatibility complex loss of heterozygosity (LOH), allele-specific mutation and measurement of expression and repression (MHC Hammer). We identified extensive variability in HLA allelic expression and pervasive HLA alternative splicing in normal lung and breast tissue. In lung TRACERx and lung and breast TCGA cohorts, 61% of lung adenocarcinoma (LUAD), 76% of lung squamous cell carcinoma (LUSC) and 35% of estrogen receptor-positive (ER+) cancers harbored class I HLA transcriptional repression, while HLA tumor-enriched alternative splicing occurred in 31%, 11% and 15% of LUAD, LUSC and ER+ cancers. Consistent with the importance of HLA dysfunction in tumor evolution, in LUADs, HLA LOH was associated with metastasis and LUAD primary tumor regions seeding a metastasis had a lower effective neoantigen burden than non-seeding regions. These data highlight the extent and importance of HLA transcriptomic disruption, including repression and alternative splicing in cancer evolution.

Cancer cell - Fibroblast crosstalk via HB-EGF, EGFR, and MAPK signaling promotes the expression of macrophage chemo-attractants in squamous cell carcinoma.

Interactions between cells in the tumor microenvironment (TME) shape cancer progression and patient prognosis. To gain insights into how the TME influences cancer outcomes, we derive gene expression signatures indicative of signaling between stromal fibroblasts and cancer cells, and demonstrate their prognostic significance in multiple and independent squamous cell carcinoma cohorts. By leveraging information within the signatures, we discover that the HB-EGF/EGFR/MAPK axis represents a hub of tumor-stroma crosstalk, promoting the expression of CSF2 and LIF and favoring the recruitment of macrophages. Together, these analyses demonstrate the utility of our approach for interrogating the extent and consequences of TME crosstalk.

Real-time estimation of immunological responses against emerging SARS-CoV-2 variants in the UK: a mathematical modelling study.

BACKGROUND: The emergence of SARS-CoV-2 variants and COVID-19 vaccination have resulted in complex exposure histories. Rapid assessment of the effects of these exposures on neutralising antibodies against SARS-CoV-2 infection is crucial for informing vaccine strategy and epidemic management. We aimed to investigate heterogeneity in individual-level and population-level antibody kinetics to emerging variants by previous SARS-CoV-2 exposure history, to examine implications for real-time estimation, and to examine the effects of vaccine-campaign timing. METHODS: Our Bayesian hierarchical model of antibody kinetics estimated neutralising-antibody trajectories against a panel of SARS-CoV-2 variants quantified with a live virus microneutralisation assay and informed by individual-level COVID-19 vaccination and SARS-CoV-2 infection histories. Antibody titre trajectories were modelled with a piecewise linear function that depended on the key biological quantities of an initial titre value, time the peak titre is reached, set-point time, and corresponding rates of increase and decrease for gradients between two timing parameters. All process parameters were estimated at both the individual level and the population level. We analysed data from participants in the University College London Hospitals-Francis Crick Institute Legacy study cohort (NCT04750356) who underwent surveillance for SARS-CoV-2 either through asymptomatic mandatory occupational health screening once per week between April 1, 2020, and May 31, 2022, or symptom-based testing between April 1, 2020, and Feb 1, 2023. People included in the Legacy study were either Crick employees or health-care workers at three London hospitals, older than 18 years, and gave written informed consent. Legacy excluded people who were unable or unwilling to give informed consent and those not employed by a qualifying institution. We segmented data to include vaccination events occurring up to 150 days before the emergence of three variants of concern: delta, BA.2, and XBB 1.5. We split the data for each wave into two categories: real-time and retrospective. The real-time dataset contained neutralising-antibody titres collected up to the date of emergence in each wave; the retrospective dataset contained all samples until the next SARS-CoV-2 exposure of each individual, whether vaccination or infection. FINDINGS: We included data from 335 participants in the delta wave analysis, 223 (67%) of whom were female and 112 (33%) of whom were male (median age 40 years, IQR 22-58); data from 385 participants in the BA.2 wave analysis, 271 (70%) of whom were female and 114 (30%) of whom were male (41 years, 22-60); and data from 248 participants in the XBB 1.5 wave analysis, 191 (77%) of whom were female, 56 (23%) of whom were male, and one (<1%) of whom preferred not to say (40 years, 21-59). Overall, we included 968 exposures (vaccinations) across 1895 serum samples in the model. For the delta wave, we estimated peak titre values as 490·0 IC50 (95% credible interval 224·3-1515·9) for people with no previous infection and as 702·4 IC50 (300·8-2322·7) for people with a previous infection before omicron; the delta wave did not include people with a previous omicron infection. For the BA.2 wave, we estimated peak titre values as 858·1 IC50 (689·8-1363·2) for people with no previous infection, 1020·7 IC50 (725·9-1722·6) for people with a previous infection before omicron, and 1422·0 IC50 (679·2-3027·3) for people with a previous omicron infection. For the XBB 1.5 wave, we estimated peak titre values as 703·2 IC50 (415·0-3197·8) for people with no previous infection, 1215·9 IC50 (511·6-7338·7) for people with a previous infection before omicron, and 1556·3 IC50 (757·2-7907·9) for people with a previous omicron infection. INTERPRETATION: Our study shows the feasibility of real-time estimation of antibody kinetics before SARS-CoV-2 variant emergence. This estimation is valuable for understanding how specific combinations of SARS-CoV-2 exposures influence antibody kinetics and for examining how COVID-19 vaccination-campaign timing could affect population-level immunity to emerging variants. FUNDING: Wellcome Trust, National Institute for Health Research University College London Hospitals Biomedical Research Centre, UK Research and Innovation, UK Medical Research Council, Francis Crick Institute, and Genotype-to-Phenotype National Virology Consortium.

An agent-based modelling framework to study growth mechanisms in EGFR-L858R mutant cell alveolar type II cells.

Mutations in the epidermal growth factor receptor (EGFR) are common in non-small cell lung cancer (NSCLC), particularly in never-smoker patients. However, these mutations are not always carcinogenic, and have recently been reported in histologically normal lung tissue from patients with and without lung cancer. To investigate the outcome of EGFR mutation in healthy lung stem cells, we grow murine alveolar type II organoids monoclonally in a three-dimensional Matrigel. Our experiments show that the EGFR-L858R mutation induces a change in organoid structure: mutated organoids display more 'budding', in comparison with non-mutant controls, which are nearly spherical. We perform on-lattice computational simulations, which suggest that this can be explained by the concentration of division among a small number of cells on the surface of the mutated organoids. We are currently unable to distinguish the cell-based mechanisms that lead to this spatial heterogeneity in growth, but suggest a number of future experiments which could be used to do so. We suggest that the likelihood of L858R-fuelled tumorigenesis is affected by whether the mutation arises in a spatial environment that allows the development of these surface protrusions. These data may have implications for cancer prevention strategies and for understanding NSCLC progression.

Replication timing alterations are associated with mutation acquisition during breast and lung cancer evolution.

During each cell cycle, the process of DNA replication timing is tightly regulated to ensure the accurate duplication of the genome. The extent and significance of alterations in this process during malignant transformation have not been extensively explored. Here, we assess the impact of altered replication timing (ART) on cancer evolution by analysing replication-timing sequencing of cancer and normal cell lines and 952 whole-genome sequenced lung and breast tumours. We find that 6%-18% of the cancer genome exhibits ART, with regions with a change from early to late replication displaying an increased mutation rate and distinct mutational signatures. Whereas regions changing from late to early replication contain genes with increased expression and present a preponderance of APOBEC3-mediated mutation clusters and associated driver mutations. We demonstrate that ART occurs relatively early during cancer evolution and that ART may have a stronger correlation with mutation acquisition than alterations in chromatin structure.

Implementing circulating tumor DNA as a prognostic biomarker in resectable non-small cell lung cancer.

Systemic treatment of resectable non-small cell lung cancer (NSCLC) is evolving with emerging neoadjuvant, perioperative, and adjuvant immunotherapy approaches. Circulating tumor DNA (ctDNA) detection at clinical diagnosis, during neoadjuvant therapy, or after resection may discern high-risk patients who might benefit from therapy escalation or switch. This Review summarizes translational implications of data supporting ctDNA-based risk determination in NSCLC and outstanding questions regarding ctDNA validity/utility as a prognostic biomarker. We discuss emerging ctDNA capabilities to refine clinical tumor-node-metastasis (TNM) staging in lung adenocarcinoma, ctDNA dynamics during neoadjuvant therapy for identifying patients deriving suboptimal benefit, and postoperative molecular residual disease (MRD) detection to escalate systemic therapy. Considering differential relapse characteristics in landmark MRD-negative/MRD-positive patients, we propose how ctDNA might integrate with pathological response data for optimal postoperative risk stratification.

Deep cell phenotyping and spatial analysis of multiplexed imaging with TRACERx-PHLEX.

The growing scale and dimensionality of multiplexed imaging require reproducible and comprehensive yet user-friendly computational pipelines. TRACERx-PHLEX performs deep learning-based cell segmentation (deep-imcyto), automated cell-type annotation (TYPEx) and interpretable spatial analysis (Spatial-PHLEX) as three independent but interoperable modules. PHLEX generates single-cell identities, cell densities within tissue compartments, marker positivity calls and spatial metrics such as cellular barrier scores, along with summary graphs and spatial visualisations. PHLEX was developed using imaging mass cytometry (IMC) in the TRACERx study, validated using published Co-detection by indexing (CODEX), IMC and orthogonal data and benchmarked against state-of-the-art approaches. We evaluated its use on different tissue types, tissue fixation conditions, image sizes and antibody panels. As PHLEX is an automated and containerised Nextflow pipeline, manual assessment, programming skills or pathology expertise are not essential. PHLEX offers an end-to-end solution in a growing field of highly multiplexed data and provides clinically relevant insights.

Mixed responses to targeted therapy driven by chromosomal instability through p53 dysfunction and genome doubling.

The phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone. The combined presence of whole genome doubling (WGD) and TP53 co-mutations leads to increased genome instability and genomic copy number aberrations in genes implicated in EGFR TKI resistance. Using mouse models and an in vitro isogenic p53-mutant model system, we provide evidence that WGD provides diverse routes to drug resistance by increasing the probability of acquiring copy-number gains or losses relative to non-WGD cells. These data provide a molecular basis for mixed tumor responses to targeted therapy, within an individual patient, with implications for therapeutic strategies.

Tumor Promoters and Opportunities for Molecular Cancer Prevention.

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.

Why do patients with cancer die?

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.

The History of Chromosomal Instability in Genome-Doubled Tumors.

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.

Representation of genomic intratumor heterogeneity in multi-region non-small cell lung cancer patient-derived xenograft models.

Patient-derived xenograft (PDX) models are widely used in cancer research. To investigate the genomic fidelity of non-small cell lung cancer PDX models, we established 48 PDX models from 22 patients enrolled in the TRACERx study. Multi-region tumor sampling increased successful PDX engraftment and most models were histologically similar to their parent tumor. Whole-exome sequencing enabled comparison of tumors and PDX models and we provide an adapted mouse reference genome for improved removal of NOD scid gamma (NSG) mouse-derived reads from sequencing data. PDX model establishment caused a genomic bottleneck, with models often representing a single tumor subclone. While distinct tumor subclones were represented in independent models from the same tumor, individual PDX models did not fully recapitulate intratumor heterogeneity. On-going genomic evolution in mice contributed modestly to the genomic distance between tumors and PDX models. Our study highlights the importance of considering primary tumor heterogeneity when using PDX models and emphasizes the benefit of comprehensive tumor sampling.

The mutagenic forces shaping the genomic landscape of lung cancer in never smokers.

Lung cancer in never smokers (LCINS) accounts for up to 25% of all lung cancers and has been associated with exposure to secondhand tobacco smoke and air pollution in observational studies. Here, we evaluate the mutagenic exposures in LCINS by examining deep whole-genome sequencing data from a large international cohort of 871 treatment-naïve LCINS recruited from 28 geographical locations within the Sherlock-Lung study. KRAS mutations were 3.8-fold more common in adenocarcinomas of never smokers from North America and Europe, while a 1.6-fold higher prevalence of EGFR and TP53 mutations was observed in adenocarcinomas from East Asia. Signature SBS40a, with unknown cause, was found in most samples and accounted for the largest proportion of single base substitutions in adenocarcinomas, being enriched in EGFR-mutated cases. Conversely, the aristolochic acid signature SBS22a was almost exclusively observed in patients from Taipei. Even though LCINS exposed to secondhand smoke had an 8.3% higher mutational burden and 5.4% shorter telomeres, passive smoking was not associated with driver mutations in cancer driver genes or the activities of individual mutational signatures. In contrast, patients from regions with high levels of air pollution were more likely to have TP53 mutations while exhibiting shorter telomeres and an increase in most types of somatic mutations, including a 3.9-fold elevation of signature SBS4 (q-value=3.1 × 10-5), previously linked mainly to tobacco smoking, and a 76% increase of clock-like signature SBS5 (q-value=5.0 × 10-5). A positive dose-response effect was observed with air pollution levels, which correlated with both a decrease in telomere length and an elevation in somatic mutations, notably attributed to signatures SBS4 and SBS5. Our results elucidate the diversity of mutational processes shaping the genomic landscape of lung cancer in never smokers.

Spatial Architecture of Myeloid and T Cells Orchestrates Immune Evasion and Clinical Outcome in Lung Cancer.

Understanding the role of the tumor microenvironment (TME) in lung cancer is critical to improving patient outcomes. We identified four histology-independent archetype TMEs in treatment-naïve early-stage lung cancer using imaging mass cytometry in the TRACERx study (n = 81 patients/198 samples/2.3 million cells). In immune-hot adenocarcinomas, spatial niches of T cells and macrophages increased with clonal neoantigen burden, whereas such an increase was observed for niches of plasma and B cells in immune-excluded squamous cell carcinomas (LUSC). Immune-low TMEs were associated with fibroblast barriers to immune infiltration. The fourth archetype, characterized by sparse lymphocytes and high tumor-associated neutrophil (TAN) infiltration, had tumor cells spatially separated from vasculature and exhibited low spatial intratumor heterogeneity. TAN-high LUSC had frequent PIK3CA mutations. TAN-high tumors harbored recently expanded and metastasis-seeding subclones and had a shorter disease-free survival independent of stage. These findings delineate genomic, immune, and physical barriers to immune surveillance and implicate neutrophil-rich TMEs in metastasis. SIGNIFICANCE: This study provides novel insights into the spatial organization of the lung cancer TME in the context of tumor immunogenicity, tumor heterogeneity, and cancer evolution. Pairing the tumor evolutionary history with the spatially resolved TME suggests mechanistic hypotheses for tumor progression and metastasis with implications for patient outcome and treatment. This article is featured in Selected Articles from This Issue, p. 897.

Cancer biomarkers: Emerging trends and clinical implications for personalized treatment.

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.

Embracing cancer complexity: Hallmarks of systemic disease.

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.

COVID-19 in non-hospitalised adults caused by either SARS-CoV-2 sub-variants Omicron BA.1, BA.2, BA.4/5 or Delta associates with similar illness duration, symptom severity and viral kinetics, irrespective of vaccination history.

BACKGROUND: SARS-CoV-2 variant Omicron rapidly evolved over 2022, causing three waves of infection due to sub-variants BA.1, BA.2 and BA.4/5. We sought to characterise symptoms and viral loads over the course of COVID-19 infection with these sub-variants in otherwise-healthy, vaccinated, non-hospitalised adults, and compared data to infections with the preceding Delta variant of concern (VOC). METHODS: In a prospective, observational cohort study, healthy vaccinated UK adults who reported a positive polymerase chain reaction (PCR) or lateral flow test, self-swabbed on alternate weekdays until day 10. We compared participant-reported symptoms and viral load trajectories between infections caused by VOCs Delta and Omicron (sub-variants BA.1, BA.2 or BA.4/5), and tested for relationships between vaccine dose, symptoms and PCR cycle threshold (Ct) as a proxy for viral load using Chi-squared (χ2) and Wilcoxon tests. RESULTS: 563 infection episodes were reported among 491 participants. Across infection episodes, there was little variation in symptom burden (4 [IQR 3-5] symptoms) and duration (8 [IQR 6-11] days). Whilst symptom profiles differed among infections caused by Delta compared to Omicron sub-variants, symptom profiles were similar between Omicron sub-variants. Anosmia was reported more frequently in Delta infections after 2 doses compared with Omicron sub-variant infections after 3 doses, for example: 42% (25/60) of participants with Delta infection compared to 9% (6/67) with Omicron BA.4/5 (χ2 P < 0.001; OR 7.3 [95% CI 2.7-19.4]). Fever was less common with Delta (20/60 participants; 33%) than Omicron BA.4/5 (39/67; 58%; χ2 P = 0.008; OR 0.4 [CI 0.2-0.7]). Amongst infections with an Omicron sub-variants, symptoms of coryza, fatigue, cough and myalgia predominated. Viral load trajectories and peaks did not differ between Delta, and Omicron, irrespective of symptom severity (including asymptomatic participants), VOC or vaccination status. PCR Ct values were negatively associated with time since vaccination in participants infected with BA.1 (ß = -0.05 (CI -0.10-0.01); P = 0.031); however, this trend was not observed in BA.2 or BA.4/5 infections. CONCLUSION: Our study emphasises both the changing symptom profile of COVID-19 infections in the Omicron era, and ongoing transmission risk of Omicron sub-variants in vaccinated adults. TRIAL REGISTRATION: NCT04750356.

Research autopsy programmes in oncology: shared experience from 14 centres across the world.

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.