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.

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 non-small cell lung cancer metastases in TRACERx.

Metastatic disease is responsible for the majority of cancer-related deaths1. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse.

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.

Immunogenomics of Colorectal Cancer Response to Checkpoint Blockade: Analysis of the KEYNOTE 177 Trial and Validation Cohorts.

BACKGROUND & AIMS: Colorectal cancer (CRC) shows variable response to immune checkpoint blockade, which can only partially be explained by high tumor mutational burden (TMB). We conducted an integrated study of the cancer tissue and associated tumor microenvironment (TME) from patients treated with pembrolizumab (KEYNOTE 177 clinical trial) or nivolumab to dissect the cellular and molecular determinants of response to anti- programmed cell death 1 (PD1) immunotherapy. METHODS: We selected multiple regions per tumor showing variable T-cell infiltration for a total of 738 regions from 29 patients, divided into discovery and validation cohorts. We performed multiregional whole-exome and RNA sequencing of the tumor cells and integrated these with T-cell receptor sequencing, high-dimensional imaging mass cytometry, detection of programmed death-ligand 1 (PDL1) interaction in situ, multiplexed immunofluorescence, and computational spatial analysis of the TME. RESULTS: In hypermutated CRCs, response to anti-PD1 immunotherapy was not associated with TMB but with high clonality of immunogenic mutations, clonally expanded T cells, low activation of Wnt signaling, deregulation of the interferon gamma pathway, and active immune escape mechanisms. Responsive hypermutated CRCs were also rich in cytotoxic and proliferating PD1+CD8 T cells interacting with PDL1+ antigen-presenting macrophages. CONCLUSIONS: Our study clarified the limits of TMB as a predictor of response of CRC to anti-PD1 immunotherapy. It identified a population of antigen-presenting macrophages interacting with CD8 T cells that consistently segregate with response. We therefore concluded that anti-PD1 agents release the PD1-PDL1 interaction between CD8 T cells and macrophages to promote cytotoxic antitumor activity.

Clonal architecture in mesothelioma is prognostic and shapes the tumour microenvironment.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.

Pervasive chromosomal instability and karyotype order in tumour evolution.

Chromosomal instability in cancer consists of dynamic changes to the number and structure of chromosomes1,2. The resulting diversity in somatic copy number alterations (SCNAs) may provide the variation necessary for tumour evolution1,3,4. Here we use multi-sample phasing and SCNA analysis of 1,421 samples from 394 tumours across 22 tumour types to show that continuous chromosomal instability results in pervasive SCNA heterogeneity. Parallel evolutionary events, which cause disruption in the same genes (such as BCL9, MCL1, ARNT (also known as HIF1B), TERT and MYC) within separate subclones, were present in 37% of tumours. Most recurrent losses probably occurred before whole-genome doubling, that was found as a clonal event in 49% of tumours. However, loss of heterozygosity at the human leukocyte antigen (HLA) locus and loss of chromosome 8p to a single haploid copy recurred at substantial subclonal frequencies, even in tumours with whole-genome doubling, indicating ongoing karyotype remodelling. Focal amplifications that affected chromosomes 1q21 (which encompasses BCL9, MCL1 and ARNT), 5p15.33 (TERT), 11q13.3 (CCND1), 19q12 (CCNE1) and 8q24.1 (MYC) were frequently subclonal yet appeared to be clonal within single samples. Analysis of an independent series of 1,024 metastatic samples revealed that 13 focal SCNAs were enriched in metastatic samples, including gains in chromosome 8q24.1 (encompassing MYC) in clear cell renal cell carcinoma and chromosome 11q13.3 (encompassing CCND1) in HER2+ breast cancer. Chromosomal instability may enable the continuous selection of SCNAs, which are established as ordered events that often occur in parallel, throughout tumour evolution.

The T cell differentiation landscape is shaped by tumour mutations in lung cancer.

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.

Publisher Correction: Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: A clonal expression biomarker associates with lung cancer mortality.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Interplay between whole-genome doubling and the accumulation of deleterious alterations in cancer evolution.

Whole-genome doubling (WGD) is a prevalent event in cancer, involving a doubling of the entire chromosome complement. However, despite its prevalence and prognostic relevance, the evolutionary selection pressures for WGD in cancer have not been investigated. Here, we combine evolutionary simulations with an analysis of cancer sequencing data to explore WGD during cancer evolution. Simulations suggest that WGD can be selected to mitigate the irreversible, ratchet-like, accumulation of deleterious somatic alterations, provided that they occur at a sufficiently high rate. Consistent with this, we observe an enrichment for WGD in tumor types with extensive loss of heterozygosity, including lung squamous cell carcinoma and triple-negative breast cancers, and we find evidence for negative selection against homozygous loss of essential genes before, but not after, WGD. Finally, we demonstrate that loss of heterozygosity and temporal dissection of mutations can be exploited to identify novel tumor suppressor genes and to obtain a deeper characterization of known cancer genes.

Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse.

Approximately 50% of patients with early-stage non-small-cell lung cancer (NSCLC) who undergo surgery with curative intent will relapse within 5 years1,2. Detection of circulating tumor cells (CTCs) at the time of surgery may represent a tool to identify patients at higher risk of recurrence for whom more frequent monitoring is advised. Here we asked whether CellSearch-detected pulmonary venous CTCs (PV-CTCs) at surgical resection of early-stage NSCLC represent subclones responsible for subsequent disease relapse. PV-CTCs were detected in 48% of 100 patients enrolled into the TRACERx study3, were associated with lung-cancer-specific relapse and remained an independent predictor of relapse in multivariate analysis adjusted for tumor stage. In a case study, genomic profiling of single PV-CTCs collected at surgery revealed higher mutation overlap with metastasis detected 10 months later (91%) than with the primary tumor (79%), suggesting that early-disseminating PV-CTCs were responsible for disease relapse. Together, PV-CTC enumeration and genomic profiling highlight the potential of PV-CTCs as early predictors of NSCLC recurrence after surgery. However, the limited sensitivity of PV-CTCs in predicting relapse suggests that further studies using a larger, independent cohort are warranted to confirm and better define the potential clinical utility of PV-CTCs in early-stage NSCLC.

A clonal expression biomarker associates with lung cancer mortality.

An aim of molecular biomarkers is to stratify patients with cancer into disease subtypes predictive of outcome, improving diagnostic precision beyond clinical descriptors such as tumor stage1. Transcriptomic intratumor heterogeneity (RNA-ITH) has been shown to confound existing expression-based biomarkers across multiple cancer types2-6. Here, we analyze multi-region whole-exome and RNA sequencing data for 156 tumor regions from 48 patients enrolled in the TRACERx study to explore and control for RNA-ITH in non-small cell lung cancer. We find that chromosomal instability is a major driver of RNA-ITH, and existing prognostic gene expression signatures are vulnerable to tumor sampling bias. To address this, we identify genes expressed homogeneously within individual tumors that encode expression modules of cancer cell proliferation and are often driven by DNA copy-number gains selected early in tumor evolution. Clonal transcriptomic biomarkers overcome tumor sampling bias, associate with survival independent of clinicopathological risk factors, and may provide a general strategy to refine biomarker design across cancer types.

Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer.

Somatic mutations together with immunoediting drive extensive heterogeneity within non-small-cell lung cancer (NSCLC). Herein we examine heterogeneity of the T cell antigen receptor (TCR) repertoire. The number of TCR sequences selectively expanded in tumors varies within and between tumors and correlates with the number of nonsynonymous mutations. Expanded TCRs can be subdivided into TCRs found in all tumor regions (ubiquitous) and those present in a subset of regions (regional). The number of ubiquitous and regional TCRs correlates with the number of ubiquitous and regional nonsynonymous mutations, respectively. Expanded TCRs form part of clusters of TCRs of similar sequence, suggestive of a spatially constrained antigen-driven process. CD8+ tumor-infiltrating lymphocytes harboring ubiquitous TCRs display a dysfunctional tissue-resident phenotype. Ubiquitous TCRs are preferentially detected in the blood at the time of tumor resection as compared to routine follow-up. These findings highlight a noninvasive method to identify and track relevant tumor-reactive TCRs for use in adoptive T cell immunotherapy.

Neoantigen-directed immune escape in lung cancer evolution.

The interplay between an evolving cancer and a dynamic immune microenvironment remains unclear. Here we analyse 258 regions from 88 early-stage, untreated non-small-cell lung cancers using RNA sequencing and histopathology-assessed tumour-infiltrating lymphocyte estimates. Immune infiltration varied both between and within tumours, with different mechanisms of neoantigen presentation dysfunction enriched in distinct immune microenvironments. Sparsely infiltrated tumours exhibited a waning of neoantigen editing during tumour evolution, indicative of historical immune editing, or copy-number loss of previously clonal neoantigens. Immune-infiltrated tumour regions exhibited ongoing immunoediting, with either loss of heterozygosity in human leukocyte antigens or depletion of expressed neoantigens. We identified promoter hypermethylation of genes that contain neoantigenic mutations as an epigenetic mechanism of immunoediting. Our results suggest that the immune microenvironment exerts a strong selection pressure in early-stage, untreated non-small-cell lung cancers that produces multiple routes to immune evasion, which are clinically relevant and forecast poor disease-free survival.

Computational Methods for Analysis of Tumor Clonality and Evolutionary History.

Cancer is an evolutionary process. Recent advances in sequencing technologies have allowed us to investigate intratumor heterogeneity at the single nucleotide level. Here, we describe computational methods that use sequencing data to identify genetically distinct tumor subclones and reconstruct tumor evolutionary histories.