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.

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.

Supplemental Digital Content is available in the text. Checkpoint inhibitors (CPIs) have demonstrated a heterogenous spectrum of response and disease progression that may not be fully captured by conventional response criteria, such as a limited degree of progression, known as oligoprogression, which could benefit from local treatment. We retrospectively analyzed data from all patients diagnosed with metastatic melanoma, who received CPI between January 2006 and March 2018 at Royal Marsden. We enrolled 36 patients who experienced progression in a maximum of 3 metastatic sites, after achieving disease control from therapy with CPI, and were radically treated with the locoregional approach. We carried out Kaplan-Meier analysis to obtain progression free-survival post-first oligoprogression (PFS-PO1), overall survival (OS) post-first oligoprogression, and OS estimates. The median time to oligoprogression from the start of CPI was 12 months. At a median follow-up of 34 months, the median PFS-PO1 was 32 months, with 50% of patients not progressed at the time of the data cutoff. The median OS-post-first oligoprogression was not reached. At a median follow-up of 52 months (from the first cycle of CPI), the median OS was not reached, with 75% of patients alive at the time of analysis. Univariate and multivariate analyses demonstrated that baseline American Joint Committee on Cancer stage IV M1a or M1b is associated with a longer PFS-PO1 compared with stage M1c or M1d. We observed that local therapy for oligoprogression after CPI can result in durable disease control, suggesting that locoregional treatment should be considered in patients being treated with immunotherapy. However, prospective evaluation, perhaps in randomized trials, is needed.

Background Immune-related diarrhoea/colitis (ir-D/C) is a common adverse event of immune checkpoint inhibitor (ICI) therapy. Guidelines recommend corticosteroid (CS) treatment; however, the average treatment duration for ir-D/C remains poorly defined. Methods All advanced melanoma patients treated with ICI therapy at the Royal Marsden Hospital between 2011 and 2016 were reviewed to identify ir-D/C cases alongside clinical variables. Results 117 any-grade ir-D/C episodes occurred in 109 (21%) patients out of a total of 519 patients treated (ipilimumab=77 episodes, anti-PD1=17 (nivolumab or pembrolizumab), ipilimumab and nivolumab=23 (ipi+nivo)) (seven patients had ir-D/C more than once on different lines of treatment) and >/=grade 3 ir-D/C occurred most frequently (63/519 patients (12%) vs 29/519 (5%) grade 1, and 25/519 (5%) grade 2). Median onset (days) of all-grade ir-D/C after starting ICI therapy was 41 for ipilimumab (IQR 24 to 59, n=77), 91 for anti-PD1 (IQR 46 to 355, n=17) and 45 for ipi+nivo (IQR 24 to 67, n=23). In 71/117 (61%) patients, ir-D/C episodes were treated with CS (17% grade 2; 79% grade 3/4): 54 being steroid-responsive; 17 being steroid-refractory and received additional anti-tumor necrosis factor (TNF) treatment. Median grade 3 ir-D/C CS duration was similar across treatments, averaging 58 days. Median overall CS duration (days) was longer in the grade 3/4 D/C steroid-refractory group (94 vs 45 days). Infection developed in 11/71 (15%) CS recipients and in 6/17 (35%) anti-TNF recipients. In 65/117 (55%) patients, ir-D/C episodes were investigated with flexible sigmoidoscopy. Of these patients, 38/65 (58%) had macroscopic colitis and 12/65 (18%) had microscopic colitis. The steroid-refractory group had more macroscopic changes, 13/17 (76%), than the steroid-responsive group, 22/41 (54%). Conclusion Rates of grade 3 ir-D/C were higher than reported in clinical trials. The 58-day median duration of CS therapy for grade 3 ir-D/C places a significant number of patients at risk of complications. We demonstrate that microscopic colitis is an important subgroup, advocating biopsies in ir-D/C even with macroscopically normal bowel.

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 40,000 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 standard operating procedure (SOP) for high-throughput SARS- CoV-2 detection by RT-LAMP in 25 minutes that is robust, reliable, repeatable, sensitive, specific, and inexpensive.

Purpose Ipilimumab, a monoclonal antibody inhibiting CLTA-4, is an established treatment in metastatic melanoma, either alone or in combination with nivolumab, and results in immune mediated adverse events, including endocrinopathy. Hypophysitis is one of the most common endocrine abnormalities. An early recognition of hypophysitis may prevent life threatening consequences of hypopituitarism; therefore, biomarkers to predict which patients will develop hypophysitis would have clinical utility. Recent studies suggested that a decline in TSH may serve as an early marker of IH. This study was aimed at assessing the utility of thyroid function tests in predicting development of hypophysitis. Methods A retrospective cohort study was performed for all patients ( n  = 308) treated with ipilimumab either as a monotherapy or in combination with nivolumab for advanced melanoma at the Royal Marsden Hospital from 2010 to 2016. Thyroid function tests, other pituitary function tests and Pituitary MRIs were used to identify those with hypophysitis. Results and conclusions Ipilimumab-induced hypophysitis (IH) was diagnosed in 25 patients (8.15%). A decline in TSH was observed in hypophysitis cohort during the first three cycles but it did not reach statistical significance ( P  = 0.053). A significant fall in FT4 ( P  < 0.001), TSH index ( P  < 0.001) and standardised TSH index ( P  < 0.001) prior to cycles 3 and 4 in hypophysitis cohort was observed. TSH is not useful in predicting development of IH. FT4, TSH index and standardised TSH index may be valuable but a high index of clinical suspicion remains paramount in early detection of hypophysitis.

Although thousands of solid tumors have been sequenced to date, a fundamental under-sampling bias is inherent in current methodologies. This is caused by a tissue sample input of fixed dimensions (e.g., 6 mm biopsy), which becomes grossly under-powered as tumor volume scales. Here, we demonstrate representative sequencing (Rep-Seq) as a new method to achieve unbiased tumor tissue sampling. Rep-Seq uses fixed residual tumor material, which is homogenized and subjected to next-generation sequencing. Analysis of intratumor tumor mutation burden (TMB) variability shows a high level of misclassification using current single-biopsy methods, with 20% of lung and 52% of bladder tumors having at least one biopsy with high TMB but low clonal TMB overall. Misclassification rates by contrast are reduced to 2% (lung) and 4% (bladder) when a more representative sampling methodology is used. Rep-Seq offers an improved sampling protocol for tumor profiling, with significant potential for improved clinical utility and more accurate deconvolution of clonal structure.

Motivation As multi-region, time-series, and single cell sequencing data become more widely available, it is becoming clear that certain tumors share evolutionary characteristics with others. In the last few years, several computational methods have been developed with the goal of inferring the subclonal composition and evolutionary history of tumors from tumor biopsy sequencing data. However, the phylogenetic trees that they report differ significantly between tumors (even those with similar characteristics). Results In this paper, we present a novel combinatorial optimization method, CONETT, for detection of recurrent tumor evolution trajectories. Our method constructs a consensus tree of conserved evolutionary trajectories based on the information about temporal order of alteration events in a set of tumors. We apply our method to previously published datasets of 100 clear-cell renal cell carcinoma and 99 non-small-cell lung cancer patients and identify both conserved trajectories that were reported in the original studies, as well as new trajectories. Availability CONETT is implemented in C++ and available at https://github.com/ehodzic/CONETT.

The expectation for survival in patients with advanced melanoma now exceeds 50% at 5 years in patients treated with first-line combination ipilimumab and nivolumab, despite this regimen being associated with substantial toxicity. We discuss the latest updates from the Checkmate-067 study, framing the role of this combination in practice today.