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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.

In the version of this article initially published, the list of members and affiliations for The TRACERx Consortium was provided as a Supplementary Note. The list should have been provided in the online version. Also, an incorrect version of Fig. 2b was published. The correct version is provided below. The errors have been corrected in the HTML and PDF versions of the article.

Introduction: Studies of cancer evolution have relied on archival tissue surplus to diagnostic requirements from living patients obtained in early stage disease and less commonly relapsed/metastatic disease. PEACE (Posthumous Evaluation of Advanced Cancer Environment) is a national research autopsy study aiming to understand the biological processes driving metastatic disease and cancer evolution. Experimental procedures: Patients recruited into the national TRACERx (TRAcking Cancer Evolution through therapy (Rx)) lung study who subsequently developed metastatic disease were enrolled into PEACE. Here we present a cohort of 15 TRACERx/PEACE patients in whom multi-region tumor sampling was performed at diagnosis +/- relapse and at autopsy. Fresh frozen tissue was subjected to whole-exome sequencing (mean depth 390) and germline DNA from blood was used for reference. Single nucleotide variants (SNVs) were identified using VarScan2 and the subclonal composition of each tumor was inferred using PyClone and used to reconstruct phylogenetic trees. Summary of data: Disease progression was associated with increased genomic complexity. Different patterns of progression occurred; monoclonal/monophyletic, polyclonal/monophyletic and polyclonal/polyphyletic dissemination. Both early and late divergence relative to the last clonal sweep were observed. Putative drivers were found to occur both clonally and subclonally. SNVs and copy number aberrations were used to reconstruct migration patterns. Conclusions: Preliminary analysis in this cohort demonstrates increasing genomic complexity with disease progression and variation in patterns of metastatic dissemination. We are yet to establish the impact of the tumor and immune microenvironment on such patterns as well as clinical presentation and outcome. Ongoing PEACE analysis includes study of the somatic copy number landscape and the characterisation of subclones that seed metastases. Patient & sample characteristicsN (%)Total patients15Median age (IQR)73 (65 - 80)Female sex (%)6 (40%)Histological subtypeAdenocarcinoma7 (47%)Squamous cell carcinoma5 (33%)Large cell carcinoma2 (13%)Adenosquamous carcinoma1 (7%)Treatment receivedRadiotherapy8 (53%)Chemotherapy6 (40%)Immunotherapy5 (33%)Targeted therapy4 (27%)Smoking statusCurrent smoker2 (13%)Ex-smoker12 (80%)Never smoker1 (7%)Total metastases sampled289Median samples per patient (range)19 (5 - 41)Number of samples per patient5-104 (27%)11-205 (33%)>206 (40%)Number of tissue types sampled17 Citation Format: Ariana Huebner, Sonya Hessey, Cristina Naceur-Lombardelli, Mita Akther, Selvaraju Veeriah, Maise Al Bakir, David Moore, Simone Zaccaria, Nicholas McGranahan, Charles Swanton, Mariam Jamal-Hanjani, TRACERx and PEACE consortium. Lung cancer evolutionary trajectories in PEACE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3123.

Frameshift insertion/deletions (fs-indels) are an infrequent but potentially highly immunogenic mutation subtype. Although fs-indel transcripts are susceptible to degradation through the non-sense mediated decay (NMD) pathway, we hypothesise that some fs-indels escape degradation and lead to an increased abundance of tumor specific neoantigens, that are highly distinct from self. We analysed matched DNA and RNA sequencing data from TCGA, and five separate melanoma cohorts treated with immunotherapy. Using allele-specific expression analysis we show that expressed fs-indels were enriched in genomic positions predicted to escape NMD, and associated with higher protein expression, consistent with degradation escape (“NMD-escape”). Across four independent cohorts, fs-indel NMD-escape mutations were found to be significantly associated with clinical benefit to checkpoint inhibitor (CPI) therapy (Pmeta=0.0039), a stronger association than either nsSNV (Pmeta=0.073) or fs-indel (Pmeta=0.064) count. NMD-escape mutations were additionally shown to have independent predictive power in the “low-TMB” setting, and may serve as a biomarker to rescue patients judged ineligible for CPI based on overall TMB, but still with a high chance of response (low-TMB cohort: NMD-escape-positive % clinical benefit=53%, NMD-escape-negative % clinical benefit=16%, P=0.0098). Furthermore, in an adoptive cell therapy (ACT) treated cohort, NMD-escape mutation count was the most significant biomarker associated with clinical benefit (P=0.021). Analysis of functional T-cell reactivity screens from recent personalized vaccine and CPI studies shows direct evidence of fs-indel derived neoantigens eliciting patient anti-tumor immune response (n=15). We additionally observe a subset of fs-indel mutations, with highly elongated neo open reading frames, which are found to be significantly enriched for immunogenic reactivity in these patient studies (P=0.0032). Finally, consistent with the potency of NMD-escape derived neo-antigens and ongoing immune-editing, NMD-escape fs-indels appear to be under negative selective pressure in untreated TCGA cases. Given the strongly immunogenic potential, and relatively rare nature of NMD-escape fs-indels, these alterations may be attractive candidates in immunotherapy biomarker optimisation and neoantigen ACT or vaccine strategies.

Introduction Minimal residual disease (MRD) detection in solid tumors describes isolation of circulating tumor DNA (ctDNA) molecules in plasma following definitive treatment of a cancer. Detection of MRD following surgical tumor excision categorizes patients as high risk for disease recurrence. Establishing an MRD approach to treating early-stage NSCLC will facilitate escalation of standard of care (SoC) treatment only in patients destined to relapse from their cancer and overcome challenges associated with conventional adjuvant drug-trial design. Here, we present data from the lung TRACERx study where patients with early-stage NSCLC underwent phylogenetic ctDNA profiling following resection. Methods Patient specific anchored-multiplex PCR (AMP) enrichment panels were generated for 78 lung TRACERx patients who underwent surgery for stage I-III NSCLC; 608 plasma samples were analyzed. Extensive patient-specific cfDNA enrichment panels targeted a median of 196 (range 72 to 482) clonal and subclonal variants detected in primary tumor tissue by multi-region exome sequencing. A novel MRD-caller controlled and estimated background sequencing error to maximize ctDNA detection at low mutant allele frequencies (MAFs). Analytical validation experiments benchmarked assay performance. Results Analytical validation of a 50-variant AMP-MRD assay demonstrated a sensitivity of 89% for mutant DNA at a MAF of 0.008% (with 25ng of DNA input into the assay), specificity was 100% experimentally and 99.9% (95% CI: 99.67 to 99.99%) modelled in-silico. 45 patients suffered relapse of their primary NSCLC; ctDNA was detected at or before clinical relapse in 37 of 45 patients. In these 37 patients the median ctDNA lead-time (time from ctDNA detection to clinical relapse) was 151 days (range 0 to 984 days) and the median time to relapse from surgery was 413 days (range 41 to 1242 days). In 10 of 10 patients who developed second primary cancers during follow-up no ctDNA was detected, reflecting specificity of the MRD assay toward the primary tumor. In 23 patients who remained relapse-free during a median of 1184 days of study follow-up, ctDNA was detected in 1 of 199 time-points analyzed. Analysis of SoC adjuvant surveillance imaging (CT, PET-CT or MRI, 220 encounters) revealed examples of MRD positive patients where SoC radiological surveillance was negative for impending relapse. Through application of large cfDNA enrichment panels targeting up to 483 variants per patient we observed dynamic changes in clonal composition and copy-number status prior to NSCLC relapse, categorized relapse as monoclonal or polyclonal and identified distinct subclonal dynamics during systemic intervention for disease recurrence. Conclusions ctDNA is an adjuvant biomarker capable of both detecting MRD following surgery and defining the clonality of relapsing disease. These data pave the way for clinical trials predicated on escalation of adjuvant standard of care in NSCLC patients who exhibit MRD positive status following surgery. Citation Format: Chris Abbosh, Alexander Frankell, Aaron Garnett, Thomas Harrison, Morgan Weichert, Abel Licon, Selvaraju Veeriah, Bob Daber, Mike Moreau, Adrian Chesh, Kevin Litchfield, Emilia Lim, Daniel Cooke, Clare Puttick, Maise Al Bakir, Fabio Gomes, Akshay Patel, Lizi Manzano, Ariana Huebner, Nicolas Carey, Joan Riley, Paula Roberts, Todd Druley, Jacqui A. Shaw, Nicholas McGranahan, Mariam Jamal-Hanjani, Nicolai Birkbak, Josh Stahl, Charles Swanton, Lung TRACERx consortium. Phylogenetic tracking and minimal residual disease detection using ctDNA in early-stage NSCLC: A lung TRACERx study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT023.

Summary 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 −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.

e18701Background: Intensive care units (ICU) are increasingly managing cancer patients (pts) with life-threatening disease and treatment-related complications. 5% of cancer pts require ICU admissio...

Nature 545, 446–451 (2017); doi:10.1038/nature22364 For 6 of the 96 patients included in this Article (patients CRUK0014, CRUK0030, CRUK0048, CRUK0059, CRUK0096 and CRUK0097) incorrect tumour volumetric data and positron emission tomography (PET) tumour background ratio (TBR) data were analysed. This error occurred because of the incorrect assignment of patient identifiers during the anonymization mandated by the independent review board of pre-operative computed tomography (CT) scans belonging to these patients.

Introduction Epithelial ovarian cancer (EOC) is a heterogeneous condition with poor survival outcomes. The genetics of hereditary and sporadic ovarian cancers will be covered and its implications to management and future research are discussed. Sources of data Key recent published literature. Areas of agreement Both genetic and environmental factors play a role in the development of EOC. Most EOCs develop sporadically and are divided into low-grade/genetically stable type I tumours and high-grade/genetically unstable type II tumours. The commonest hereditary syndromes are hereditary breast ovarian cancer syndrome (HBOC-BRCA mutations) and Lynch syndrome (DNA mismatch repair mutations). Areas of controversy The different histological types of EOC may not solely originate from the ovary but from the fallopian tube and endometriosis deposits; there is increasing evidence to support this. Growing points Our understanding of the genetics and frequencies of mutations in ovarian cancer is expanding. The proportion of heritable EOC is larger than previously estimated and not all patients have a clear family history for this. Mutations in genes involving the downstream BRCA signalling pathway have recently been implicated in HBOC. TP53 mutations are the single most commonly identified mutations in aggressive sporadic high-grade serous carcinomas, affecting essentially 100% of such tumours. Furthermore, there is increasing recognition that the different histological sub-types need to be treated as separate entities. Areas for timely research Given how heterogeneous 'ovarian' cancer is, trials into new drugs should report responses for the different histo-/geno-types rather than simply using staging. Although the effect of new drugs such as poly(ADP-ribose) polymerase inhibitors are being investigated in ovarian cancer, there is still a need to develop targeted therapies-especially to tackle mutations in PI3 K pathway, RAS pathway and TP53.

5570 Background: Non-epithelial malignancies of the ovary account for