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.

Summary Checkpoint inhibitors (CPIs) augment adaptive immunity. Systematic pan-tumor analyses may reveal the relative importance of tumor-cell-intrinsic and microenvironmental features underpinning CPI sensitization. Here, we collated whole-exome and transcriptomic data for >1,000 CPI-treated patients across seven tumor types, utilizing standardized bioinformatics workflows and clinical outcome criteria to validate multivariable predictors of CPI sensitization. Clonal tumor mutation burden (TMB) was the strongest predictor of CPI response, followed by total TMB and CXCL9 expression. Subclonal TMB, somatic copy alteration burden, and histocompatibility leukocyte antigen (HLA) evolutionary divergence failed to attain pan-cancer significance. Dinucleotide variants were identified as a source of immunogenic epitopes associated with radical amino acid substitutions and enhanced peptide hydrophobicity/immunogenicity. Copy-number analysis revealed two additional determinants of CPI outcome supported by prior functional evidence: 9q34 (TRAF2) loss associated with response and CCND1 amplification associated with resistance. Finally, single-cell RNA sequencing (RNA-seq) of clonal neoantigen-reactive CD8 tumor-infiltrating lymphocytes (TILs), combined with bulk RNA-seq analysis of CPI-responding tumors, identified CCR5 and CXCL13 as T-cell-intrinsic markers of CPI sensitivity.

Close to 74,000 cases of renal cell carcinoma (RCC) are diagnosed each year in the United States. The past 2 decades have shown great developments in surgical techniques, targeted therapy and immunotherapy agents, and longer complete response rates. However, without a global cure, there is still room for further advancement in improving patient care in this space. To address some of the gaps restricting this progress, the Kidney Cancer Association brought together a group of 27 specialists across the areas of clinical care, research, industry, and advocacy at the inaugural "Think Tank: Coalition for a Cure" session. Topics addressed included screening, imaging, rarer RCC subtypes, combination drug therapy options, and patient response. This commentary summarizes the discussion of these topics and their respective clinical challenges, along with a proposal of projects for collaboration in overcoming those needs and making a greater impact on care for patients with RCC moving forward.

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.

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. Chromosomal instability enables the continuous selection of somatic copy number alterations, which are established as ordered events that often occur in parallel, throughout tumour evolution and metastasis.

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.

Adoptive transfer of tumor infiltrating lymphocytes (TIL) has generated objective clinical responses in patients with advanced metastatic cancers. Therapeutic exploitation of neoantigens as targets can potentially lead to safer and more effective treatment modalities with reduced toxicities. The Achilles Therapeutics trial NCT03517917 enabled the acquisition of matched tumor specimens and peripheral blood samples from patients undergoing routine surgery and facilitated the development of the proprietary VELOSTM manufacturing process, generating a personalized clonal neoantigen specific T cell product. An in-depth characterization of T cells expanded with the VELOSTM process was performed and compared to a standard TIL product. Samples were obtained from patients with primary NSCLC or metastatic melanoma. TIL were expanded from tumor fragments after dissection in the presence of IL-2. Peptide pools corresponding to the clonal mutations that were identified using the PELEUSTM bioinformatics platform were used to pulse dendritic cells (DC) generated from peripheral blood monocytes from each patient. Clonal neoantigen specific T cells (cNeT) were expanded using the VELOSTM process by co-culture of TIL with the peptide-pulsed autologous DC. As a comparison, TIL were expanded with a rapid expansion protocol (REP-TIL) in the presence of allogeneic feeders, anti-CD3 antibody and high-dose IL-2. Intracellular cytokine staining was performed following rechallenge with individual peptide pools encoding the clonal mutations. Single peptide reactivities were identified using ELISPOT and extended flow cytometric analysis of markers associated with T cell fitness or dysfunction was performed to phenotypically characterize the cNeT, TIL and REP-TIL. Analysis of the immune cell composition showed that cNeT, TIL and REP-TIL have similar CD3+ T cell content (median cNeT 90.2%, TIL 87.3%, REP-TIL 95%, n=6) and are composed of CD4+ and CD8+ T cells (median CD4:CD8 ratio- cNeT 11.1, TIL 2.03 and REP-TIL 4.7, n=6). cNeT showed superior clonal neoantigen specificity compared to TIL or REP-TIL. The proportion of CD3+ T cells responding to clonal neoantigen rechallenge was increased in cNeT (median 24.3%) compared to TIL (median 0.6%) and REP-TIL (median 1.8%) (n=5). The VELOSTM process incorporating the PELEUSTM platform for prediction of clonal neoantigens generates T cell products enriched for clonal neoantigen reactivities and superior phenotypic characteristics compared to conventional TIL. The VELOSTM process is currently being used to manufacture cNeT for two first-in-human studies including NSCLC and melanoma patients (NCT04032847, NCT03997474). Ethical approval: The samples for the study were collected under an ethically approved protocol (NCT03517917). Citation Format: Eleni Kotsiou, Tie Zheng Hou, Joseph Robinson, Sonal Varsani, Theres Oakes, Pablo D. Becker, Shreenal Patel, Jennine Mootien, Andrew Craig, Jane Robertson, Edward Samuel, James Reading, Lyra Del Rosario, Andrew Haynes, Samra Turajlic, Farah Islam, David Lawrence, Mariam Jamal-Hanjani, Martin Foster, Sergio A. Quezada, Katy Newton. Next generation clonal neoantigen targeting T cells, generated using the PELEUSTM bioinformatics platform and the VELOSTM manufacturing method show superior reactivity and phenotypic characteristics than classical TIL products [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 875.