Intra-tumour genetic heterogeneity (ITH) fuels cancer evolution. The role of clonal diversity and genetic complexity in the progression of clear-cell renal cell carcinomas (ccRCCs) has been characterised, but the ability to predict clinically relevant evolutionary trajectories remains limited. Here, towards enhancing this ability, we investigated spatial features of clonal diversification through a combined computational modelling and experimental analysis in the TRACERx Renal study. We observe through modelling that spatial patterns of tumour growth impact the extent and trajectory of subclonal diversification. Moreover, subpopulations with high clonal diversity, and parallel evolution events, are frequently observed near the tumour margin. In-silico time-course studies further showed that budding structures on the tumour surface could indicate future steps of subclonal evolution. Such structures were evident radiologically in 15 early-stage ccRCCs, raising the possibility that spatially resolved sampling of these regions, when combined with sequencing, may enable identification of evolutionary potential in early-stage tumours.

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARS-CoV-2 detection by RT-LAMP that is robust, reliable, repeatable, specific, and inexpensive.

The ongoing pandemic of SARS-CoV-2 calls for rapid and cost-effective methods to accurately identify infected individuals. The vast majority of patient samples is assessed for viral RNA presence by RT-qPCR. Our biomedical research institute, in collaboration between partner hospitals and an accredited clinical diagnostic laboratory, established a diagnostic testing pipeline that has reported on more than 252,000 RT-qPCR results since its commencement at the beginning of April 2020. However, due to ongoing demand and competition for critical resources, alternative testing strategies were sought. In this work, we present a clinically-validated procedure for high-throughput SARSCoV-2 detection by RT-LAMP in 25 minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive. Open Peer Review Reviewer Status AWAITING PEER REVIEW Any reports and responses or comments on the article can be found at the end of the article. Page 1 of 13 Wellcome Open Research 2021, 6:9 Last updated: 12 FEB 2021

Dedifferentiation and acquisition of chromosomal instability in renal cell carcinoma portends dismal prognosis and aggressive clinical behavior. However, the absence of reliable experimental models dramatically impacts the understanding of mechanisms underlying malignant progression. Here we established an in vivo genetic platform to rapidly generate somatic mosaic genetically engineerd immune-competent mouse models of renal tumors, recapitulating the genomic and phenotypic features of these malignancies. Leveraging somatic chromosomal engineering, we demonstrated that ablation of the murine locus syntenic to human 9p21 drives the rapid expansion of aggressive mesenchymal clones with prominent metastatic behavior, characterized by early emergence of chromosomal instability, whole-genome duplication, and conserved patterns of aneuploidy. This model of punctuated equilibrium provides a remarkable example of cross-species convergent evolution. Significance To better understand the role of 9p21 in malignant progression, we generated a somatic mosaic GEMM of renal cancer, capturing the histological, genomic and evolutionary features of human disease. With this technology we demonstrated a critica role of 9p21 loss in metastatic evolution of RCC and provide a unique tool for testing new therapeutic treatments.

Checkpoint inhibitor (CPI) therapy has significantly improved overall survival for metastatic melanoma, and is now approved for use in the adjuvant setting. Modulating the immune system is recognized to cause cutaneous immune‐related adverse events (irAEs). We conducted a retrospective observational cohort study of adult patients with melanoma at our tertiary referral centre, who received CPI therapy from 2006 to March 2018. This is the single largest study of cutaneous irAEs occurring on CPI therapy in patients with melanoma to date and encompasses 12 years. The results showed that cutaneous toxicity occurs in 24% of patients but is generally manageable, with < 5% patients discontinuing treatment.

Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumour cell intrinsic and microenvironmental features underpinning CPI sensitization. Here we collated whole-exome and transcriptomic data for >1000 CPI-treated patients across eight tumor-types, utilizing standardized bioinformatics-workflows and clinical outcome-criteria to validate multivariate predictors of CPI-sensitization. Clonal-TMB was the strongest predictor of CPI response, followed by TMB and CXCL9 expression. Subclonal-TMB, somatic copy alteration burden and HLA-evolutionary divergence failed to attain significance. Discovery analysis identified two additional determinants of CPI-response supported by prior functional evidence: 9q34.3 (TRAF2) loss and CCND1 amplification, both independently validated in >1600 CPI-treated patients. We find evidence for collateral sensitivity, likely mediated through selection for CDKN2A-loss, with 9q34.3 loss as a passenger event leading to CPI-sensitization. Finally, scRNA sequencing of clonal neoantigen-reactive CD8-TILs, combined with bulk RNAseq analysis of CPI responding tumors, identified CCR5 and CXCL13 as T cell-intrinsic mediators of CPI-sensitisation.

While the genetic evolutionary features of solid tumour growth are becoming increasingly described, the spatial and physical nature of subclonal growth remains unclear. Here we utilise 102 macroscopic whole tumour images from clear cell renal cell carcinoma (ccRCC) patients, with matched genetic and phenotypic data from 756 biopsies. Utilising a digital image processing pipeline the boundaries between tumour and normal tissue were marked by a renal pathologist, and positions of boundary line and biopsy regions were extracted to X- and Y-coordinates. The coordinates were then integrated with genomic data to map exact spatial subclone locations, revealing how genetically distinct subclones grow and evolve spatially. A phenotype of advanced and more aggressive subclonal growth was present in the tumour centre, characterised by an elevated burden of somatic copy number alterations, higher necrosis, proliferation rate and Fuhrman grade. Moreover, metastasising subclones were found to preferentially originate from the tumour centre. Collectively these observations suggest a model of accelerated evolution in the tumour interior, with harsh hypoxic environmental conditions leading to heightened cellular turnover and greater opportunity for driver SCNAs to arise and expand due to selective advantage. Tumour subclone growth was found to be predominantly spatially contiguous in nature, with subclone dispersal a rare event found in two cases, which notably was associated with metastasis. In terms of genetic events, the largest subclones spatially were dominated by driver somatic copy number alterations, suggesting a large selective advantage can be conferred to subclones upon acquisition of these alterations. In conclusion, spatial dynamics is strongly associated with genomic alterations and plays an important role in tumour evolution.

Background: Clonal neoantigens are formed early in cancer evolution and have been identified as a subset of patient specific mutations that are associated with improved clinical benefit and represent great promise as targets for the next generation of T cell therapies. Developing T cell therapies that target multiple clonal neoantigens represents a unique personalized approach to treating solid cancer, as they are present on all cancer cells, minimizing the risk of tumour escape, and absent from healthy tissue, potentially eliminating off-target toxicities. Access to sequencing data from over 600 NSCLC patients enrolled in the UK TRACERx study has enabled the development of the Achilles PELEUSTM bioinformatic platform. By opening an ethically approved tissue collection study NCT03517917, enabling access to matched tumour and blood samples from patients with selected cancers, our clonal neoantigen reactive T cell (cNeT) manufacturing process and supply chain has been validated for use in clinical trials. Methods: Matched tumor and blood samples were procured at the time of routine surgery from ten patients (eight with newly diagnosed stage I-III NSCLC and two with metastatic melanoma) for at-scale GMP runs. Briefly, TIL were isolated from tumor fragments and immature dendritic cells (DCs) generated from whole blood, prior to cryopreservation as intermediate products. Patient-specific clonal neoantigens were predicted using our proprietary PELEUSTM bioinformatic platform, enabling the manufacture of synthetic peptide masterpools to be used for the enrichment of cNeT in the VELOSTM manufacturing process. Co-culture of pre-expanded TIL and patient DCs loaded with clonal neoantigen peptides drives the selective expansion of cNeT, eliminating the requirement for high non-physiological levels of IL-2. Results: Here we present the successful scaled GMP production of cNeT from both primary and metastatic tumors using the VELOSTM manufacturing process in ten patients. All final products met QC release criteria and were composed of both CD4+ and CD8+ T cells. Extensive characterization of T cell responses showed cNeT exhibited functional responses determined by cytokine secretion following re-challenge, and specificity in response to clonal neoantigen peptides. Peptide deconvolution of masterpools identified multiple single T cell clone reactivities to clonal neoantigens in the final product. Conclusions: The VELOSTM process incorporating the PELEUSTM bioinformatic platform for prediction of clonal neoantigens is a novel platform for generating personalized T cell products directed at multiple cancer clonal neoantigen targets and has the potential to be utilized across a variety of solid tumors. This study demonstrates the feasibility of generating cNeT for the treatment of both advanced NSCLC and recurrent or metastatic melanoma and supported the successful regulatory approval in two first-in-human studies (NCT04032847 and NCT03997474) which opened in the UK in 2019. Citation Format: Henrieta Fraser, Rebecca Pike, Sarah Thirkell, Asiya Arshad, Sam Jide-Banwo, Hollie Bartley, Evi Rologi, Michal Pruchniak, Shreenal Patel, Jennine Mootien, Jane Robertson, Andrew Craig, Max Salm, Katy Newton, Luke Goodsell, Fong Chan, Gareth Wilson, Stephen Frenk, Iraj Ali, Karl Peggs, Mark W. Lowdell, Lyra Del Rosio, Andrew Hayes, Samra Turajlic, Farah Islam, David Lawrence, Mariam Jamal-Hanjani, Martin D. Forster, Edward Samuel. The development of a personalized autologous clonal neoantigen T cell therapy for the treatment of solid cancer using the VELOSTM manufacturing platform generates highly potent and reactive CD8+ and CD4+ T cells for clinical use [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 CT054.