Histologically normal human tissues accumulate significant mutational burden with age. The extent and spectra of mutagenesis are comparable both in rapidly proliferating and post-mitotic tissues and ...in stem cells compared with their differentiated progeny. Some of these mutations provide increased fitness, giving rise to clones which, at times, can replace the entire surface area of tissues. Compared with cancer, somatic mutations in histologically normal tissues are primarily single-nucleotide variations. Interestingly though, the presence of these mutations and positive clonal selection in isolation remains a poor indicator of potential future cancer transformation in solid tissues. Common clonally expanded mutations in histologically normal tissues also do not always represent the most frequent early mutations in cancers of corresponding tissues, indicating differences in selection pressures. Preliminary evidence implies that stroma and immune system co-evolve with age, which may impact selection dynamics. In this review, we will explore the mutational landscape of histologically normal and premalignant human somatic tissues in detail and discuss cell-intrinsic and environmental factors that can determine the fate of positively selected mutations within them. Precisely pinpointing these determinants of cancer transformation would aid development of early cancer interventional and prevention strategies.
In gastric and gastroesophageal adenocarcinoma, circulating tumor DNA identifies molecular drivers missed using routine tumor biopsy, can refine patient selection for targeted therapy, and may be ...prognostic following surgical resection. However, lack of standardization could impede adoption of this technology as a clinically useful tool; prospective validation in clinical trials is required.
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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.
Metastatic disease is responsible for the majority of cancer-related deaths
. 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.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Intratumour heterogeneity (ITH) fuels lung cancer evolution, which leads to immune evasion and resistance to therapy
. 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 study
. 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.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)
. 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 elusive
. 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 adenocarcinoma
. 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.
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Lung cancer is the leading cause of cancer-associated mortality worldwide
. 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.
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Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy
. 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 study
. 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.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Abstract
Background: Cancer evolution occurs though a Darwinian process of natural selection. Selective sweeps occur in most human tumours which obscure early, unsuccessful lineages. In addition, ...analysis of bulk samples has limited phylogenies to SNVs and Indels, due to methodological difficulties in bulk clonal deconvolution of copy number aberrations (CNAs). Using single cell whole genome sequencing we construct phylogenies of CNAs for murine models of the early stages in lung adenocarcinoma evolution and define features of unsuccessful lineages not yet obscured by full selective sweeps. Methods: Non-invasive tumour tissue was homogenised from EGFRL858R and EGFRL858R/TP53 knock out (KO) mice and subject to 0.1X single cell WGS. Bowtie and AnneuFinder were used to align FASTQ files and call CNAs respectively. A strict quality control step was step applied using read depth and coverage variability, removing 15% of cells. MEDDIC was used to construct phylogenetic trees and ancestral CN states at each node. These ancestral CN states were translated into distinct CN events in the evolutionary history of each tumour. Results: We reconstructed tumour CNA phylogenies using scWGS to determine phylogenetic relationships and time CN events. CNAs in these data were far more heterogenous than in previously published human studies. We noted small populations of progenitor cells which branched off early in evolutionary time, containing far fewer copy number aberrations and which were not genome doubled. These populations allow us to more accurately time CN events such as would be obscured after a full selective sweep. Whole chromosomal events were significantly more likely to occur earlier in evolutionary history than intra-chromosomal events (Wilcoxon test, P < 1e-16, OR = 2.24) and chromosomal gains were also significantly more common at the earlier stages of tumour evolution (Wilcoxon test, P < 1e-16, OR = 1.44). Within whole chromosomal events, gains were significantly more enriched for early timing (Wilcoxon test, P < 1e-16, OR = 7.11). Progenitor cell populations also contained a small number of additional events, which were usually unique to a single cell. We reasoned these events may represent evolutionary dead ends. We found these events were highly enriched for losses in TP53 wild type tumours (Wilcoxon test, P < 1e-16, OR = 11.9) but much less so in TP53 mutant tumours (Wilcoxon test, P = 1e-6, OR = 1.14) suggesting early negative selection against loss of heterozygosity, particularly when TP53 is intact. Discussion: The observed depletion of intrachromosomal events may be caused by a more intact DNA damage response system which is therefore less permissive to chromosomal breaks. Heterozygous losses may be negatively selected due to resultant decreased dosage of a large numbers of genes, upon which the cells become less reliant as cancer progresses. These data are consistent with a recent report of spontaneous CNAs in culture of normal hTERT lines which have a strong preference for gains in a TP53 WT context but not upon TP53 KO.
Citation Format: Alexander M. Frankell, Sebastian Hobor, Maise Al-bakir, Eva Grönroos, Charles Swanton. Single cell whole genome sequencing reveals the dynamics of copy number instability at the earliest stages of cancer evolution abstract. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-018.
The immune microenvironment influences tumour evolution and can be both prognostic and predict response to immunotherapy
. However, measurements of tumour infiltrating lymphocytes (TILs) are limited ...by a shortage of appropriate data. Whole-exome sequencing (WES) of DNA is frequently performed to calculate tumour mutational burden and identify actionable mutations. Here we develop T cell exome TREC tool (T cell ExTRECT), a method for estimation of T cell fraction from WES samples using a signal from T cell receptor excision circle (TREC) loss during V(D)J recombination of the T cell receptor-α gene (TCRA (also known as TRA)). TCRA T cell fraction correlates with orthogonal TIL estimates and is agnostic to sample type. Blood TCRA T cell fraction is higher in females than in males and correlates with both tumour immune infiltrate and presence of bacterial sequencing reads. Tumour TCRA T cell fraction is prognostic in lung adenocarcinoma. Using a meta-analysis of tumours treated with immunotherapy, we show that tumour TCRA T cell fraction predicts immunotherapy response, providing value beyond measuring tumour mutational burden. Applying T cell ExTRECT to a multi-sample pan-cancer cohort reveals a high diversity of the degree of immune infiltration within tumours. Subclonal loss of 12q24.31-32, encompassing SPPL3, is associated with reduced TCRA T cell fraction. T cell ExTRECT provides a cost-effective technique to characterize immune infiltrate alongside somatic changes.
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