Decoding the interplay between genetic and non-genetic drivers of metastasis.

Metastasis is a multistep process by which cancer cells break away from their original location and spread to distant organs, and is responsible for the vast majority of cancer-related deaths. Preventing early metastatic dissemination would revolutionize the ability to fight cancer. Unfortunately, the relatively poor understanding of the molecular underpinnings of metastasis has hampered the development of effective anti-metastatic drugs. Although it is now accepted that disseminating tumour cells need to acquire multiple competencies to face the many obstacles they encounter before reaching their metastatic site(s), whether these competencies are acquired through an accumulation of metastasis-specific genetic alterations and/or non-genetic events is often debated. Here we review a growing body of literature highlighting the importance of both genetic and non-genetic reprogramming events during the metastatic cascade, and discuss how genetic and non-genetic processes act in concert to confer metastatic competencies. We also describe how recent technological advances, and in particular the advent of single-cell multi-omics and barcoding approaches, will help to better elucidate the cross-talk between genetic and non-genetic mechanisms of metastasis and ultimately inform innovative paths for the early detection and interception of this lethal process.

Genomic-transcriptomic evolution in lung cancer and metastasis.

Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy1. Here, using paired whole-exome and RNA sequencing data, we investigate intratumour transcriptomic diversity in 354 non-small cell lung cancer tumours from 347 out of the first 421 patients prospectively recruited into the TRACERx study2,3. Analyses of 947 tumour regions, representing both primary and metastatic disease, alongside 96 tumour-adjacent normal tissue samples implicate the transcriptome as a major source of phenotypic variation. Gene expression levels and ITH relate to patterns of positive and negative selection during tumour evolution. We observe frequent copy number-independent allele-specific expression that is linked to epigenomic dysfunction. Allele-specific expression can also result in genomic-transcriptomic parallel evolution, which converges on cancer gene disruption. We extract signatures of RNA single-base substitutions and link their aetiology to the activity of the RNA-editing enzymes ADAR and APOBEC3A, thereby revealing otherwise undetected ongoing APOBEC activity in tumours. Characterizing the transcriptomes of primary-metastatic tumour pairs, we combine multiple machine-learning approaches that leverage genomic and transcriptomic variables to link metastasis-seeding potential to the evolutionary context of mutations and increased proliferation within primary tumour regions. These results highlight the interplay between the genome and transcriptome in influencing ITH, lung cancer evolution and metastasis.

The evolution of lung cancer and impact of subclonal selection in TRACERx.

Lung cancer is the leading cause of cancer-associated mortality worldwide1. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy.

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

Lung adenocarcinoma promotion by air pollutants.

A complete understanding of how exposure to environmental substances promotes cancer formation is lacking. More than 70 years ago, tumorigenesis was proposed to occur in a two-step process: an initiating step that induces mutations in healthy cells, followed by a promoter step that triggers cancer development1. Here we propose that environmental particulate matter measuring ≤2.5 µm (PM2.5), known to be associated with lung cancer risk, promotes lung cancer by acting on cells that harbour pre-existing oncogenic mutations in healthy lung tissue. Focusing on EGFR-driven lung cancer, which is more common in never-smokers or light smokers, we found a significant association between PM2.5 levels and the incidence of lung cancer for 32,957 EGFR-driven lung cancer cases in four within-country cohorts. Functional mouse models revealed that air pollutants cause an influx of macrophages into the lung and release of interleukin-1ß. This process results in a progenitor-like cell state within EGFR mutant lung alveolar type II epithelial cells that fuels tumorigenesis. Ultradeep mutational profiling of histologically normal lung tissue from 295 individuals across 3 clinical cohorts revealed oncogenic EGFR and KRAS driver mutations in 18% and 53% of healthy tissue samples, respectively. These findings collectively support a tumour-promoting role for  PM2.5 air pollutants  and provide impetus for public health policy initiatives to address air pollution to reduce disease burden.

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.

Clinical implications of heterogeneity in PD-L1 immunohistochemical detection in hepatocellular carcinoma: the Blueprint-HCC study.

Programmed cell death ligand-1 immunohistochemical detection (PD-L1 IHC) is a putative predictor of response to PD-1/PD-L1-targeted checkpoint inhibitors. However, there is no gold standard assay in hepatocellular carcinoma (HCC). We evaluated 5 PD-L1 IHC assay platforms (E1LN3, 28-8, 22c3, SP263 and SP142) in 100 HCCs reporting PD-L1 expression in malignant (M) and tumour-infiltrating immune cells (TICs) and non-tumorous cirrhotic tissues (NTICs). We found substantial inter-assay heterogeneity in detecting PD-L1 expression in M (R2 = 0.080-0.921), TICs (Cohen's κ = 0.175-0.396) and NTICs (κ = 0.004-0.505). Such diversity may impact on the reliability and reproducibility of PD-L1 IHC assays as a predictor of response to immune checkpoint inhibitors.

Age-related macular degeneration: genome-wide association studies to translation.

In recent years, genome-wide association studies (GWAS), which are able to analyze the contribution to disease of genetic variations that are common within a population, have attracted considerable investment. Despite identifying genetic variants for many conditions, they have been criticized for yielding data with minimal clinical utility. However, in this regard, age-related macular degeneration (AMD), the most common form of blindness in the Western world, is a striking exception. Through GWAS, common genetic variants at a number of loci have been discovered. Two loci in particular, including genes of the complement cascade on chromosome 1 and the ARMS2/HTRA1 genes on chromosome 10, have been shown to convey significantly increased susceptibility to developing AMD. Today, although it is possible to screen individuals for a genetic predisposition to the disease, effective interventional strategies for those at risk of developing AMD are scarce. Ongoing research in this area is nonetheless promising. After providing brief overviews of AMD and common disease genetics, we outline the main recent advances in the understanding of AMD, particularly those made through GWAS. Finally, the true merit of these findings and their current and potential translational value is examined.Genet Med 18 4, 283-289.

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.

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.

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance.

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.

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.

ACT-Discover: identifying karyotype heterogeneity in pancreatic cancer evolution using ctDNA.

BACKGROUND: Liquid biopsies and the dynamic tracking of somatic mutations within circulating tumour DNA (ctDNA) can provide insight into the dynamics of cancer evolution and the intra-tumour heterogeneity that fuels treatment resistance. However, identifying and tracking dynamic changes in somatic copy number alterations (SCNAs), which have been associated with poor outcome and metastasis, using ctDNA is challenging. Pancreatic adenocarcinoma is a disease which has been considered to harbour early punctuated events in its evolution, leading to an early fitness peak, with minimal further subclonal evolution. METHODS: To interrogate the role of SCNAs in pancreatic adenocarcinoma cancer evolution, we applied whole-exome sequencing of 55 longitudinal cell-free DNA (cfDNA) samples taken from 24 patients (including 8 from whom a patient-derived xenograft (PDX) was derived) with metastatic disease prospectively recruited into a clinical trial. We developed a method, Aneuploidy in Circulating Tumour DNA (ACT-Discover), that leverages haplotype phasing of paired tumour biopsies or PDXs to identify SCNAs in cfDNA with greater sensitivity. RESULTS: SCNAs were observed within 28 of 47 evaluable cfDNA samples. Of these events, 30% could only be identified by harnessing the haplotype-aware approach leveraged in ACT-Discover. The exceptional purity of PDX tumours enabled near-complete phasing of genomic regions in allelic imbalance, highlighting an important auxiliary function of PDXs. Finally, although the classical model of pancreatic cancer evolution emphasises the importance of early, homogenous somatic events as a key requirement for cancer development, ACT-Discover identified substantial heterogeneity of SCNAs, including parallel focal and arm-level events, affecting different parental alleles within individual tumours. Indeed, ongoing acquisition of SCNAs was identified within tumours throughout the disease course, including within an untreated metastatic tumour. CONCLUSIONS: This work demonstrates the power of haplotype phasing to study genomic variation in cfDNA samples and reveals undiscovered intra-tumour heterogeneity with important scientific and clinical implications. Implementation of ACT-Discover could lead to important insights from existing cohorts or underpin future prospective studies seeking to characterise the landscape of tumour evolution through liquid biopsy.

Evolutionary characterization of lung adenocarcinoma morphology in TRACERx.

Lung adenocarcinomas (LUADs) display a broad histological spectrum from low-grade lepidic tumors through to mid-grade acinar and papillary and high-grade solid, cribriform and micropapillary tumors. How morphology reflects tumor evolution and disease progression is poorly understood. Whole-exome sequencing data generated from 805 primary tumor regions and 121 paired metastatic samples across 248 LUADs from the TRACERx 421 cohort, together with RNA-sequencing data from 463 primary tumor regions, were integrated with detailed whole-tumor and regional histopathological analysis. Tumors with predominantly high-grade patterns showed increased chromosomal complexity, with higher burden of loss of heterozygosity and subclonal somatic copy number alterations. Individual regions in predominantly high-grade pattern tumors exhibited higher proliferation and lower clonal diversity, potentially reflecting large recent subclonal expansions. Co-occurrence of truncal loss of chromosomes 3p and 3q was enriched in predominantly low-/mid-grade tumors, while purely undifferentiated solid-pattern tumors had a higher frequency of truncal arm or focal 3q gains and SMARCA4 gene alterations compared with mixed-pattern tumors with a solid component, suggesting distinct evolutionary trajectories. Clonal evolution analysis revealed that tumors tend to evolve toward higher-grade patterns. The presence of micropapillary pattern and 'tumor spread through air spaces' were associated with intrathoracic recurrence, in contrast to the presence of solid/cribriform patterns, necrosis and preoperative circulating tumor DNA detection, which were associated with extra-thoracic recurrence. These data provide insights into the relationship between LUAD morphology, the underlying evolutionary genomic landscape, and clinical and anatomical relapse risk.

Body composition and lung cancer-associated cachexia in TRACERx.

Cancer-associated cachexia (CAC) is a major contributor to morbidity and mortality in individuals with non-small cell lung cancer. Key features of CAC include alterations in body composition and body weight. Here, we explore the association between body composition and body weight with survival and delineate potential biological processes and mediators that contribute to the development of CAC. Computed tomography-based body composition analysis of 651 individuals in the TRACERx (TRAcking non-small cell lung Cancer Evolution through therapy (Rx)) study suggested that individuals in the bottom 20th percentile of the distribution of skeletal muscle or adipose tissue area at the time of lung cancer diagnosis, had significantly shorter lung cancer-specific survival and overall survival. This finding was validated in 420 individuals in the independent Boston Lung Cancer Study. Individuals classified as having developed CAC according to one or more features at relapse encompassing loss of adipose or muscle tissue, or body mass index-adjusted weight loss were found to have distinct tumor genomic and transcriptomic profiles compared with individuals who did not develop such features. Primary non-small cell lung cancers from individuals who developed CAC were characterized by enrichment of inflammatory signaling and epithelial-mesenchymal transitional pathways, and differentially expressed genes upregulated in these tumors included cancer-testis antigen MAGEA6 and matrix metalloproteinases, such as ADAMTS3. In an exploratory proteomic analysis of circulating putative mediators of cachexia performed in a subset of 110 individuals from TRACERx, a significant association between circulating GDF15 and loss of body weight, skeletal muscle and adipose tissue was identified at relapse, supporting the potential therapeutic relevance of targeting GDF15 in the management of CAC.

Genetic and non-genetic clonal diversity in cancer evolution.

The observation and analysis of intra-tumour heterogeneity (ITH), particularly in genomic studies, has advanced our understanding of the evolutionary forces that shape cancer growth and development. However, only a subset of the variation observed in a single tumour will have an impact on cancer evolution, highlighting the need to distinguish between functional and non-functional ITH. Emerging studies highlight a role for the cancer epigenome, transcriptome and immune microenvironment in functional ITH. Here, we consider the importance of both genetic and non-genetic ITH and their role in tumour evolution, and present the rationale for a broad research focus beyond the cancer genome. Systems-biology analytical approaches will be necessary to outline the scale and importance of functional ITH. By allowing a deeper understanding of tumour evolution this will, in time, encourage development of novel therapies and improve outcomes for patients.