APOBEC3 as a driver of genetic intratumor heterogeneity.

Our recent study revealed that APOBEC3B is upregulated during the preinvasive stages of non-small cell lung cancer and breast cancer. In addition to its role in mediating single nucleotide variants, we propose that APOBEC3 promotes copy number intratumor heterogeneity prior to invasion, providing a substrate for cancer evolution.

Quantifying the impact of immunotherapy on RNA dynamics in cancer.

BACKGROUND: Checkpoint inhibitor (CPI) immunotherapies have provided durable clinical responses across a range of solid tumor types for some patients with cancer. Nonetheless, response rates to CPI vary greatly between cancer types. Resolving intratumor transcriptomic changes induced by CPI may improve our understanding of the mechanisms of sensitivity and resistance. METHODS: We assembled a cohort of longitudinal pre-therapy and on-therapy samples from 174 patients treated with CPI across six cancer types by leveraging transcriptomic sequencing data from five studies. RESULTS: Meta-analyses of published RNA markers revealed an on-therapy pattern of immune reinvigoration in patients with breast cancer, which was not discernible pre-therapy, providing biological insight into the impact of CPI on the breast cancer immune microenvironment. We identified 98 breast cancer-specific correlates of CPI response, including 13 genes which are known IO targets, such as toll-like receptors TLR1, TLR4, and TLR8, that could hold potential as combination targets for patients with breast cancer receiving CPI treatment. Furthermore, we demonstrate that a subset of response genes identified in breast cancer are already highly expressed pre-therapy in melanoma, and additionally we establish divergent RNA dynamics between breast cancer and melanoma following CPI treatment, which may suggest distinct immune microenvironments between the two cancer types. CONCLUSIONS: Overall, delineating longitudinal RNA dynamics following CPI therapy sheds light on the mechanisms underlying diverging response trajectories, and identifies putative targets for combination therapy.

Individualized tumor-informed circulating tumor DNA analysis for postoperative monitoring of non-small cell lung cancer.

We report a personalized tumor-informed technology, Patient-specific pROgnostic and Potential tHErapeutic marker Tracking (PROPHET) using deep sequencing of 50 patient-specific variants to detect molecular residual disease (MRD) with a limit of detection of 0.004%. PROPHET and state-of-the-art fixed-panel assays were applied to 760 plasma samples from 181 prospectively enrolled early stage non-small cell lung cancer patients. PROPHET shows higher sensitivity of 45% at baseline with circulating tumor DNA (ctDNA). It outperforms fixed-panel assays in prognostic analysis and demonstrates a median lead-time of 299 days to radiologically confirmed recurrence. Personalized non-canonical variants account for 98.2% with prognostic effects similar to canonical variants. The proposed tumor-node-metastasis-blood (TNMB) classification surpasses TNM staging for prognostic prediction at the decision point of adjuvant treatment. PROPHET shows potential to evaluate the effect of adjuvant therapy and serve as an arbiter of the equivocal radiological diagnosis. These findings highlight the potential advantages of personalized cancer techniques in MRD detection.

Individualized dynamic methylation-based analysis of cell-free DNA in postoperative monitoring of lung cancer.

BACKGROUND: The feasibility of DNA methylation-based assays in detecting minimal residual disease (MRD) and postoperative monitoring remains unestablished. We aim to investigate the dynamic characteristics of cancer-related methylation signals and the feasibility of methylation-based MRD detection in surgical lung cancer patients. METHODS: Matched tumor, tumor-adjacent tissues, and longitudinal blood samples from a cohort (MEDAL) were analyzed by ultra-deep targeted sequencing and bisulfite sequencing. A tumor-informed methylation-based MRD (timMRD) was employed to evaluate the methylation status of each blood sample. Survival analysis was performed in the MEDAL cohort (n = 195) and validated in an independent cohort (DYNAMIC, n = 36). RESULTS: Tumor-informed methylation status enabled an accurate recurrence risk assessment better than the tumor-naïve methylation approach. Baseline timMRD-scores were positively correlated with tumor burden, invasiveness, and the existence and abundance of somatic mutations. Patients with higher timMRD-scores at postoperative time-points demonstrated significantly shorter disease-free survival in the MEDAL cohort (HR: 3.08, 95% CI: 1.48-6.42; P = 0.002) and the independent DYNAMIC cohort (HR: 2.80, 95% CI: 0.96-8.20; P = 0.041). Multivariable regression analysis identified postoperative timMRD-score as an independent prognostic factor for lung cancer. Compared to tumor-informed somatic mutation status, timMRD-scores yielded better performance in identifying the relapsed patients during postoperative follow-up, including subgroups with lower tumor burden like stage I, and was more accurate among relapsed patients with baseline ctDNA-negative status. Comparing to the average lead time of ctDNA mutation, timMRD-score yielded a negative predictive value of 97.2% at 120 days prior to relapse. CONCLUSIONS: The dynamic methylation-based analysis of peripheral blood provides a promising strategy for postoperative cancer surveillance. TRIAL REGISTRATION: This study (MEDAL, MEthylation based Dynamic Analysis for Lung cancer) was registered on ClinicalTrials.gov on 08/05/2018 (NCT03634826). https://clinicaltrials.gov/ct2/show/NCT03634826 .

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.

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.

Multi-omics integrated circulating cell-free DNA genomic signatures enhanced the diagnostic performance of early-stage lung cancer and postoperative minimal residual disease.

BACKGROUND: Liquid biopsy is a promising non-invasive alternative for cancer screening and minimal residual disease (MRD) detection, although there are some concerns regarding its clinical applications. We aimed to develop an accurate detection platform based on liquid biopsy for both cancer screening and MRD detection in patients with lung cancer (LC), which is also applicable to clinical use. METHODS: We applied a modified whole-genome sequencing (WGS) -based High-performance Infrastructure For MultIomics (HIFI) method for LC screening and postoperative MRD detection by combining the hyper-co-methylated read approach and the circulating single-molecule amplification and resequencing technology (cSMART2.0). FINDINGS: For early screening of LC, the LC score model was constructed using the support vector machine, which showed sensitivity (51.8%) at high specificity (96.3%) and achieved an AUC of 0.912 in the validation set prospectively enrolled from multiple centers. The screening model achieved detection efficiency with an AUC of 0.906 in patients with lung adenocarcinoma and outperformed other clinical models in solid nodule cohort. When applied the HIFI model to real social population, a negative predictive value (NPV) of 99.92% was achieved in Chinese population. Additionally, the MRD detection rate improved significantly by combining results from WGS and cSMART2.0, with sensitivity of 73.7% at specificity of 97.3%. INTERPRETATION: In conclusion, the HIFI method is promising for diagnosis and postoperative monitoring of LC. FUNDING: This study was supported by CAMS Innovation Fund for Medical Sciences, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, Beijing Natural Science Foundation and Peking University People's Hospital.

Research Progress on Postoperative Minimal/Molecular Residual Disease Detection in Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide. Approximately 10%-50% of patients experience relapse after radical surgery, which may be attributed to the persistence of minimal/molecular residual disease (MRD). Circulating tumor DNA (ctDNA), a common liquid biopsy approach, has been demonstrated to have significant clinical merit. In this study, we review the evidence supporting the use of ctDNA for MRD detection and discuss the potential clinical applications of postoperative MRD detection, including monitoring recurrence, guiding adjuvant treatment, and driving clinical trials in lung cancer. We will also discuss the problems that prevent the routine application of ctDNA MRD detection. Multi-analyte methods and identification of specific genetic and molecular alterations, especially methylation, are effective detection strategies and show considerable prospects for future development. Interventional prospective studies based on ctDNA detection are needed to determine whether the application of postoperative MRD detection can improve the clinical outcomes of lung cancer patients, and the accuracy, sensitivity, specificity, and robustness of different detection methods still require optimization and refinement.

Spatiotemporal genomic analysis reveals distinct molecular features in recurrent stage I non-small cell lung cancers.

Stage I non-small cell lung cancer (NSCLC) presents diverse outcomes. To identify molecular features leading to tumor recurrence in early-stage NSCLC, we perform multiregional whole-exome sequencing (WES), RNA sequencing, and plasma-targeted circulating tumor DNA (ctDNA) detection analysis between recurrent and recurrent-free stage I NSCLC patients (CHN-P cohort) who had undergone R0 resection with a median 5-year follow-up time. Integrated analysis indicates that the multidimensional clinical and genomic model can stratify the prognosis of stage I NSCLC in both CHN-P and EUR-T cohorts and correlates with positive pre-surgical deep next generation sequencing (NGS) ctDNA detection. Increased genomic instability related to DNA interstrand crosslinks and double-strand break repair processes is significantly associated with early tumor relapse. This study reveals important molecular insights into stage I NSCLC and may inform clinical postoperative treatment and follow-up strategies.

Induction of APOBEC3 Exacerbates DNA Replication Stress and Chromosomal Instability in Early Breast and Lung Cancer Evolution.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non-small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. SIGNIFICANCE: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355.

A comparative analysis of the mutagenicity of platinum-containing chemotherapeutic agents reveals direct and indirect mutagenic mechanisms.

Platinum-based drugs are a mainstay of cancer chemotherapy. However, their mutagenic effect can increase tumour heterogeneity, contribute to the evolution of treatment resistance and also induce secondary malignancies. We coupled whole genome sequencing with phenotypic investigations on two cell line models to compare the magnitude and examine the mechanism of mutagenicity of cisplatin, carboplatin and oxaliplatin. Cisplatin induced significantly more base substitution mutations than carboplatin or oxaliplatin when used at equitoxic concentrations on human TK6 or chicken DT40 cells, and also induced the highest number of short insertions and deletions. The analysis of base substitution spectra revealed that all three tested platinum drugs elicit both a direct mutagenic effect at purine dinucleotides, and an indirect effect of accelerating endogenous mutagenic processes, whereas the direct mutagenic effect appeared to correlate with the level of DNA damage caused as assessed through histone H2AX phosphorylation and single-cell agarose gel electrophoresis, the indirect mutagenic effects were equal. The different mutagenicity and DNA-damaging effect of equitoxic platinum drug treatments suggest that DNA damage independent mechanisms significantly contribute to their cytotoxicity. Thus, the comparatively high mutagenicity of cisplatin should be taken into account in the design of chemotherapeutic regimens.

Author Correction: Scalable and robust SARS-CoV-2 testing in an academic center.

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

Oncogenic PIK3CA induces centrosome amplification and tolerance to genome doubling.

Mutations in PIK3CA are very frequent in cancer and lead to sustained PI3K pathway activation. The impact of acute expression of mutant PIK3CA during early stages of malignancy is unknown. Using a mouse model to activate the Pik3ca H1047R hotspot mutation in the heterozygous state from its endogenous locus, we here report that mutant Pik3ca induces centrosome amplification in cultured cells (through a pathway involving AKT, ROCK and CDK2/Cyclin E-nucleophosmin) and in mouse tissues, and increased in vitro cellular tolerance to spontaneous genome doubling. We also present evidence that the majority of PIK3CA H1047R mutations in the TCGA breast cancer cohort precede genome doubling. These previously unappreciated roles of PIK3CA mutation show that PI3K signalling can contribute to the generation of irreversible genomic changes in cancer. While this can limit the impact of PI3K-targeted therapies, these findings also open the opportunity for therapeutic approaches aimed at limiting tumour heterogeneity and evolution.

Insertion-and-deletion-derived tumour-specific neoantigens and the immunogenic phenotype: a pan-cancer analysis.

BACKGROUND: The focus of tumour-specific antigen analyses has been on single nucleotide variants (SNVs), with the contribution of small insertions and deletions (indels) less well characterised. We investigated whether the frameshift nature of indel mutations, which create novel open reading frames and a large quantity of mutagenic peptides highly distinct from self, might contribute to the immunogenic phenotype. METHODS: We analysed whole-exome sequencing data from 5777 solid tumours, spanning 19 cancer types from The Cancer Genome Atlas. We compared the proportion and number of indels across the cohort, with a subset of results replicated in two independent datasets. We assessed in-silico tumour-specific neoantigen predictions by mutation type with pan-cancer analysis, together with RNAseq profiling in renal clear cell carcinoma cases (n=392), to compare immune gene expression across patient subgroups. Associations between indel burden and treatment response were assessed across four checkpoint inhibitor datasets. FINDINGS: We observed renal cell carcinomas to have the highest proportion (0·12) and number of indel mutations across the pan-cancer cohort (p<2·2 × 10-16), more than double the median proportion of indel mutations in all other cancer types examined. Analysis of tumour-specific neoantigens showed that enrichment of indel mutations for high-affinity binders was three times that of non-synonymous SNV mutations. Furthermore, neoantigens derived from indel mutations were nine times enriched for mutant specific binding, as compared with non-synonymous SNV derived neoantigens. Immune gene expression analysis in the renal clear cell carcinoma cohort showed that the presence of mutant-specific neoantigens was associated with upregulation of antigen presentation genes, which correlated (r=0·78) with T-cell activation as measured by CD8-positive expression. Finally, analysis of checkpoint inhibitor response data revealed frameshift indel count to be significantly associated with checkpoint inhibitor response across three separate melanoma cohorts (p=4·7 × 10-4). INTERPRETATION: Renal cell carcinomas have the highest pan-cancer proportion and number of indel mutations. Evidence suggests indels are a highly immunogenic mutational class, which can trigger an increased abundance of neoantigens and greater mutant-binding specificity. FUNDING: Cancer Research UK, UK National Institute for Health Research (NIHR) at the Royal Marsden Hospital National Health Service Foundation Trust, Institute of Cancer Research and University College London Hospitals Biomedical Research Centres, the UK Medical Research Council, the Rosetrees Trust, Novo Nordisk Foundation, the Prostate Cancer Foundation, the Breast Cancer Research Foundation, the European Research Council.

Correction: The Subclonal Architecture of Metastatic Breast Cancer: Results from a Prospective Community-Based Rapid Autopsy Program "CASCADE".

[This corrects the article DOI: 10.1371/journal.pmed.1002204.].

The Subclonal Architecture of Metastatic Breast Cancer: Results from a Prospective Community-Based Rapid Autopsy Program "CASCADE".

BACKGROUND: Understanding the cancer genome is seen as a key step in improving outcomes for cancer patients. Genomic assays are emerging as a possible avenue to personalised medicine in breast cancer. However, evolution of the cancer genome during the natural history of breast cancer is largely unknown, as is the profile of disease at death. We sought to study in detail these aspects of advanced breast cancers that have resulted in lethal disease. METHODS AND FINDINGS: Three patients with oestrogen-receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer and one patient with triple negative breast cancer underwent rapid autopsy as part of an institutional prospective community-based rapid autopsy program (CASCADE). Cases represented a range of management problems in breast cancer, including late relapse after early stage disease, de novo metastatic disease, discordant disease response, and disease refractory to treatment. Between 5 and 12 metastatic sites were collected at autopsy together with available primary tumours and longitudinal metastatic biopsies taken during life. Samples underwent paired tumour-normal whole exome sequencing and single nucleotide polymorphism (SNP) arrays. Subclonal architectures were inferred by jointly analysing all samples from each patient. Mutations were validated using high depth amplicon sequencing. Between cases, there were significant differences in mutational burden, driver mutations, mutational processes, and copy number variation. Within each case, we found dramatic heterogeneity in subclonal structure from primary to metastatic disease and between metastatic sites, such that no single lesion captured the breadth of disease. Metastatic cross-seeding was found in each case, and treatment drove subclonal diversification. Subclones displayed parallel evolution of treatment resistance in some cases and apparent augmentation of key oncogenic drivers as an alternative resistance mechanism. We also observed the role of mutational processes in subclonal evolution. Limitations of this study include the potential for bias introduced by joint analysis of formalin-fixed archival specimens with fresh specimens and the difficulties in resolving subclones with whole exome sequencing. Other alterations that could define subclones such as structural variants or epigenetic modifications were not assessed. CONCLUSIONS: This study highlights various mechanisms that shape the genome of metastatic breast cancer and the value of studying advanced disease in detail. Treatment drives significant genomic heterogeneity in breast cancers which has implications for disease monitoring and treatment selection in the personalised medicine paradigm.

DNA replication stress mediates APOBEC3 family mutagenesis in breast cancer.

BACKGROUND: The APOBEC3 family of cytidine deaminases mutate the cancer genome in a range of cancer types. Although many studies have documented the downstream effects of APOBEC3 activity through next-generation sequencing, less is known about their upstream regulation. In this study, we sought to identify a molecular basis for APOBEC3 expression and activation. RESULTS: HER2 amplification and PTEN loss promote DNA replication stress and APOBEC3B activity in vitro and correlate with APOBEC3 mutagenesis in vivo. HER2-enriched breast carcinomas display evidence of elevated levels of replication stress-associated DNA damage in vivo. Chemical and cytotoxic induction of replication stress, through aphidicolin, gemcitabine, camptothecin or hydroxyurea exposure, activates transcription of APOBEC3B via an ATR/Chk1-dependent pathway in vitro. APOBEC3B activation can be attenuated through repression of oncogenic signalling, small molecule inhibition of receptor tyrosine kinase signalling and alleviation of replication stress through nucleoside supplementation. CONCLUSION: These data link oncogene, loss of tumour suppressor gene and drug-induced replication stress with APOBEC3B activity, providing new insights into how cytidine deaminase-induced mutagenesis might be activated in tumourigenesis and limited therapeutically.

Perturbed hematopoiesis in mice lacking ATMIN.

The ataxia telangiectasia mutated (ATM)-interacting protein ATMIN mediates noncanonical ATM signaling in response to oxidative and replicative stress conditions. Like ATM, ATMIN can function as a tumor suppressor in the hematopoietic system: deletion of Atmin under the control of CD19-Cre results in B-cell lymphomas in aging mice. ATM signaling is essential for lymphopoiesis and hematopoietic stem cell (HSC) function; however, little is known about the role of ATMIN in hematopoiesis. We thus sought to investigate whether the absence of ATMIN would affect primitive hematopoietic cells in an ATM-dependent or -independent manner. Apart from its role in B-cell development, we show that ATMIN has an ATM-independent function in the common myeloid progenitors (CMPs) by deletion of Atmin in the entire hematopoietic system using Vav-Cre. Despite the lack of lymphoma formation, ATMIN-deficient mice developed chronic leukopenia as a result of high levels of apoptosis in B cells and CMPs and induced a compensatory mechanism in which HSCs displayed enhanced cycling. Consequently, ATMIN-deficient HSCs showed impaired regeneration ability with the induction of the DNA oxidative stress response, especially when aged. ATMIN, therefore, has multiple roles in different cell types, and its absence results in perturbed hematopoiesis, especially during stress conditions and aging.