We describe three cases of critical acute myositis with myocarditis occurring within 22 days of each other at a single institution, all within 1 month of receiving the initial cycle of the anti-PD-1 drug pembrolizumab. Analysis of T cell receptor repertoires from peripheral blood and tissues revealed a high degree of clonal expansion and public clones between cases, with several T cell clones expanded within the skeletal muscle putatively recognizing viral epitopes. All patients had recently received a COVID-19 mRNA booster vaccine prior to treatment and were positive for SARS-CoV2 Spike antibody. In conclusion, we report a series of unusually severe myositis and myocarditis following PD-1 blockade and the COVID-19 mRNA vaccination.

We describe three cases of critical acute myositis with myocarditis occurring within 22 days of each other at a single institution, all within one month of receiving the initial cycle of the anti-PD-1 drug Pembrolizumab. Analysis of T cell receptor repertoires from peripheral blood and tissues revealed a high degree of clonal expansion and public clones between cases, with several T cell clones expanded within the skeletal muscle putatively recognizing viral epitopes. All patients had recently received a COVID-19 mRNA booster vaccine prior to treatment and were positive for SARS-CoV2 Spike antibody. In conclusion, we report a series of unusually severe myositis and myocarditis following PD-1 blockade and the COVID-19 mRNA vaccination.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementWellcome Intermediate Clinical Fellowship to BPF (no. 201488/Z/ 16/Z). RAW is funded by a Wellcome Trust Doctoral Training Fellowship (no. BST00070). The Oxford Radcliffe Biobank and Oxford Centre for Histopathology Research are supported by the University of Oxford, the Oxford CRUK Cancer Centre and the NIHR Oxford Biomedical Research Centre (Molecular Diagnostics Theme/Multimodal Pathology Subtheme), and the NIHR Cancer Research Network (CRN) Thames Valley network. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Patients provided written informed consent for the donation of tissue and access to clinical records to the Oxford Radcliffe Biobank (ethical approval 19/SC/0173, projects 16/A019, 18/A064, and 19/A114). Ethics committee/IRB of University of Oxford (CUREC) gave ethical approval for this work (CUREC-1, R80630/RE001).I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

4576 Background: Bladder Urothelial Carcinoma (BLCA) is the most common type of bladder cancer and the sixth most frequent cancer in the US. High numbers of lymphocytes colocated with tumour-associated stromal (TAS) tissue has demonstrated prognostic significance for overall survival in multiple cancers. Our goal in this study was to explore the prognostic significance of an automated score to quantify the colocalisation of lymphocytes and TAS for overall survival (OS) in BLCA patients from Haematoxylin & Eosin (H&E) stained Whole Slide Images (WSIs). Methods: Two cohorts of BLCA patients were included in this study. From the UK, a cohort of 67 BLCA patients constituted cohort A. We also evaluated our method on The Cancer Genome Atlas (TCGA) bladder cancer cohort of 453 cases, which we refer to as cohort B. We developed a two-stage method for digital quantification of lymphocytic infiltrates in TAS. First, we employed an AI algorithm to recognise and classify different regions as areas of high concentration of tumour, lymphocytes and stroma for each WSI creating a segmentation map of the different tissue types. For patients with multiple WSIs, we used the slide with the highest percentage of tumour to predict survival. This algorithm had been pre-trained on a cohort of Oral cancer and was fine-tuned using annotations from 4 BLCA WSIs, 3 from cohort A and 1 from cohort B. These WSIs were excluded from the final survival analysis. Using the segmentation maps, a statistical measure for the colocalisation of TAS and lymphocytes termed the tumour-associated stroma infiltrating lymphocytes or (TASIL) score was computed. Finally, for each cohort, data was right-censored at 10 years and the digital BLCA-TASIL (BT) score’s prognostic significance for OS was investigated by fitting a Kaplan-Meier estimator and Cox proportional hazard (PH) analysis, stratifying patients into two groups based on the BT score. In each cohort, two thirds of the data was used as the discovery set to determine the best cut-off for the TASIL Score and the remaining third was used as the validation set. Results: Our classification algorithm achieved high average F1-score of 0.88 on a held-out set of unseen data for the classification of tumour, lymphocytic and stromal regions. In cohort A, higher BT score was strongly associated with better OS ( P= 0.00906) on the unseen validation data. This significant association was also found in the validation data of cohort B ( P< 0.001). Using the BT score as the only covariate, a Cox PH model for the validation data resulted in a C-index of 0.73 for cohort A and 0.57 for cohort B, respectively. Conclusions: The digital BT score showed significant prognostic value for overall survival in both BLCA cohorts, reinforcing the findings of prior work. We intend to further validate these findings on another cohort. To the best of our knowledge, this is the first attempt to predict survival solely from H&E slides in BLCA.

AIMS:Widespread disruption of healthcare services and excess mortality not directly attributed to COVID-19 occurred between March and May 2020. We undertook the first UK multicentre study of coroners' autopsies before and during this period using postmortem reports.METHODS:We reviewed reports of non-forensic coroners' autopsies performed during the first COVID-19 lockdown (23 March to 8 May 2020), and the same period in 2018. Deaths were categorised as natural non-COVID-19, COVID-19-related, non-natural (suicide, drug and alcohol-related, traumatic, other). We provided opinion regarding whether delayed access to medical care or changes in behaviour due to lockdown were a potential factor in deaths.RESULTS:Seven centres covering nine coronial jurisdictions submitted a total of 1100 coroners' autopsies (498 in 2018, 602 in 2020). In only 54 autopsies was death attributed to COVID-19 (9%). We identified a significant increase in cases where delays in accessing medical care potentially contributed to death (10 in 2018, 44 in 2020). Lockdown was a contributing factor in a proportion of suicides (24%) and drug and alcohol-related deaths (12%).CONCLUSIONS:Postmortem reports have considerable utility in evaluating excess mortality due to healthcare and wider societal disruption during a pandemic. They provide information at an individual case level that is not available from assessment of death certification data. Detailed evaluation of coroners' autopsy reports, supported by appropriate regulatory oversight, is recommended to mitigate disruption and indirect causes of mortality in future pandemics. Maintaining access to healthcare, including substance misuse and mental health services, is an important consideration.

Indirect increases in morbidity and mortality resulting from movement restrictions imposed during the COVID-19 pandemic have been identified as a public health concern.1 Deaths registered in England and Wales exceeded the 5-year average by almost 50 000 during the first 2 months of lockdown, which started on March 23, 2020.2 Confirmed COVID-19 accounted for the majority of deaths; the remaining excess deaths (>12 000) could reflect undiagnosed COVID-19 or alternatively, deaths from unrelated conditions.

Approximately 75% of bladder cancers are non-muscle invasive (NMIBC). Of these, up to 53% of cases progress to life-threatening muscle-invasive bladder cancer (MIBC). Patients with high-grade stage T1 (HGT1) NMIBC frequently undergo radical cystectomy (RC), although this represents overtreatment for many. Identification of progressors versus non-progressors could spare unnecessary treatment. Recent studies have confirmed that urothelial carcinoma is composed of two main molecular subtypes, basal and luminal, with 12% of basal tumours exhibiting epithelial-to-mesenchymal transition (EMT). Levels of immune cell infiltration have been shown to be subtype-specific. Here, we performed immunohistochemistry (IHC) for 11 antibodies relating to molecular subtypes or EMT in 26 cases of HGT1 urothelial carcinoma cases with 6 matched samples subsequently obtained at cystectomy (n = 6; 1 muscle-invasive, 5 non-muscle-invasive; 3 = pTis, 1 = pT1, 1 = pTa) and at recurrence (n = 2, pT2). RNAScope was also conducted in a subset. Expression patterns in HGT1 specimens versus MIBC (pT2+) were examined, and correlated with disease-specific survival (DSS). Levels of stromal tumour-infiltrating lymphocytes (sTILs) were assessed manually to determine whether lymphocyte infiltration was associated with DSS and whether differences existed between HGT1 and MIBC. Molecular subtype markers demonstrated increased prognostic potential compared to the EMT markers assessed. Increased expression of the luminal markers FOXA1 and SCUBE2, were found to be significantly associated with better DFS. No EMT markers were significantly associated with DFS. In areas of non-invasive papillary urothelial carcinoma, but not invasive carcinoma, sTIL levels were found to be significantly associated with DFS. While differences were observed between HGT1 cases that progressed versus those that did not, a larger cohort study is required for validation of these findings. Taken together, an emphasis on molecular subtype markers, rather than EMT markers, may be preferable when studying biomarkers of HGT1 urothelial carcinoma in the future.

High‐quality histopathology is essential for the success of clinical trials. Histopathologists have a detailed understanding of tumour biology and mechanisms of disease, as well as practical knowledge of optimal tissue handling and logistical service requirements for study delivery, such as biomarker evaluation, tissue acquisition and turnaround times. As such, histopathologist input is essential throughout every stage of research and clinical trials, from concept development and study design to trial delivery, analysis and dissemination of results. Patient recruitment to trials takes place among all healthcare settings, meaning that histopathologists make an invaluable contribution to clinical trials as part of their routine day‐to‐day work that often goes unrecognised. More complex evaluation of surgical specimens in the neoadjuvant setting and ever‐expanding minimum data sets add to the workload of every histopathologist, not just academic pathologists in tertiary centres. This is occurring against a backdrop of increasing workload pressures and a worldwide shortage of histopathologists and biomedical scientists. Providing essential histopathology support for trials at grassroots level requires funding for adequate resources including histopathologist time, education and training, biomedical scientist and administrative support and greater recognition of the contribution made by histopathology. This paper will discuss the many ways in which histopathologists are involved in clinical trials and the challenges faced in meeting the additional demands posed by trial participation and potential ways to address this, with a special emphasis on the UK model and the Cellular–Molecular Pathology Initiative (CM‐Path).

Event Abstract Back to Event Divergent prognostic significance of diagnostic biopsy stromal immune infiltrate assessment in bladder cancer treated with radical cystectomy versus radical radiotherapy Robert Pell1, Anne Kiltie1*, Selina Bhattarai2, Edward Ottley1 and Christiana Kartsonaki3 1 Department of Oncology, Medical Sciences Division, University of Oxford, United Kingdom 2 Leeds Teaching Hospitals NHS Trust, United Kingdom 3 Nuffield Department of Population Health, Medical Sciences Division, University of Oxford, United Kingdom Introduction In solid tumours, identification of high percentage coverage of stromal tissue by mononuclear immune cells has demonstrated independent prognostic significance for disease-free and overall survival. Methods We evaluated percentage stromal tissue coverage by stromal tumour infiltrating lymphocytes (sTILs) within an existing tissue microarray constructed from diagnostic biopsies tissue of patients who subsequently received radical radiotherapy or cystectomy for high grade invasive bladder cancer. A total of 226 patients treated between 1995 and 2005 were evaluated but 55 were excluded due to insufficient tissue within the TMA core to assess stromal tissue coverage. A total of 61 cystectomy cases and 110 cases radiotherapy (RT) cases were identified, including 48 RT cases treated between 1995 and 2002 (discovery cohort) and 62 treated between 2002 and 2005 (validation cohort). A reproducible percentage sTIL coverage value was taken as a low/high cut off within the cohort. Its association with disease-specific survival was assessed in each cohort using a Cox proportional hazards model, adjusting for age. Results The median age of the cystectomy cohort at diagnosis was 70; the median age of the RT cohort 78. Five-year disease-specific survival for the cystectomy, RT discovery (1995-2002) and RT validation (2002-2005) cohorts were 52%, 51% and 57% respectively: these observations are in line with existing published datasets. High sTILs within the diagnostic biopsy were associated with significantly improved disease-specific survival in patients undergoing cystectomy (HR 2.58, 95% CI 1.10-6.03, p=0.029, adjusting for age and age^2). No significant association was found between sTILs within the diagnostic biopsy and survival in either radiotherapy cohort. Conclusions We provide evidence for differing prognostic utility of sTILs within a diagnostic bladder tumour biopsy according to definitive treatment. Age-related immune senescence and/or immune modulation of the response to distant metastatic disease by recurrent primary tumour are possible explanations for the observation. Keywords: Bladder cancer, Cystectomy, Radiotherapy, Histology, Immune infiltrates, Prognosic factors Conference: Bladder Cancer Translational Research Meeting, London, United Kingdom, 29 Mar - 29 Mar, 2019. Presentation Type: Poster Topic: Optimisation of diagnostic pathways Citation: Pell R, Kiltie A, Bhattarai S, Ottley E and Kartsonaki C (2019). Divergent prognostic significance of diagnostic biopsy stromal immune infiltrate assessment in bladder cancer treated with radical cystectomy versus radical radiotherapy. Front. Oncol. Conference Abstract: Bladder Cancer Translational Research Meeting. doi: 10.3389/conf.fonc.2019.01.00002 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Feb 2019; Published Online: 27 Sep 2019. * Correspondence: Prof. Anne Kiltie, Department of Oncology, Medical Sciences Division, University of Oxford, Oxford, England, OX3 7DQ, United Kingdom, anne.kiltie@oncology.ox.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Robert Pell Anne Kiltie Selina Bhattarai Edward Ottley Christiana Kartsonaki Google Robert Pell Anne Kiltie Selina Bhattarai Edward Ottley Christiana Kartsonaki Google Scholar Robert Pell Anne Kiltie Selina Bhattarai Edward Ottley Christiana Kartsonaki PubMed Robert Pell Anne Kiltie Selina Bhattarai Edward Ottley Christiana Kartsonaki Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Digital pathology and image analysis potentially provide greater accuracy, reproducibility and standardisation of pathology‐based trial entry criteria and endpoints, alongside extracting new insights from both existing and novel features. Image analysis has great potential to identify, extract and quantify features in greater detail in comparison to pathologist assessment, which may produce improved prediction models or perform tasks beyond manual capability. In this article, we provide an overview of the utility of such technologies in clinical trials and provide a discussion of the potential applications, current challenges, limitations and remaining unanswered questions that require addressing prior to routine adoption in such studies. We reiterate the value of central review of pathology in clinical trials, and discuss inherent logistical, cost and performance advantages of using a digital approach. The current and emerging regulatory landscape is outlined. The role of digital platforms and remote learning to improve the training and performance of clinical trial pathologists is discussed. The impact of image analysis on quantitative tissue morphometrics in key areas such as standardisation of immunohistochemical stain interpretation, assessment of tumour cellularity prior to molecular analytical applications and the assessment of novel histological features is described. The standardisation of digital image production, establishment of criteria for digital pathology use in pre‐clinical and clinical studies, establishment of performance criteria for image analysis algorithms and liaison with regulatory bodies to facilitate incorporation of image analysis applications into clinical practice are key issues to be addressed to improve digital pathology incorporation into clinical trials.

AimsTo evaluate if a deep learning algorithm can be trained to identify tumour-infiltrating lymphocytes (TILs) in tissue samples of testicular germ cell tumours and to assess whether the TIL counts correlate with relapse status of the patient.MethodsTILs were manually annotated in 259 tumour regions from 28 whole-slide images (WSIs) of H&E-stained tissue samples. A deep learning algorithm was trained on half of the regions and tested on the other half. The algorithm was further applied to larger areas of tumour WSIs from 89 patients and correlated with clinicopathological data.ResultsA correlation coefficient of 0.89 was achieved when comparing the algorithm with the manual TIL count in the test set of images in which TILs were present (n=47). In the WSI regions from the 89 patient samples, the median TIL density was 1009/mm2. In seminomas, none of the relapsed patients belonged to the highest TIL density tertile (>2011/mm2). TIL quantifications performed visually by three pathologists on the same tumours were not significantly associated with outcome. The average interobserver agreement between the pathologists when assigning a patient into TIL tertiles was 0.32 (Kappa test) compared with 0.35 between the algorithm and the experts, respectively. A higher TIL density was associated with a lower clinical tumour stage, seminoma histology and lack of lymphovascular invasion.ConclusionsDeep learning–based image analysis can be used for detecting TILs in testicular germ cell cancer more objectively and it has potential for use as a prognostic marker for disease relapse.

e12023 Background: We are observing increasing numbers of patients with advanced breast cancer and peritoneal metastases. There are few published data regarding the prognosis, clinical characteristics, and management of such individuals. Methods: The electronic imaging database at Charing Cross Hospital was searched for the terms ’breast,’ ’cancer or tumor,’ ’peritoneal,’ and ’ascites’ from 2000–2008. Those with confirmed peritoneal disease from breast cancer, as described on ultrasound or staging CT reports with a clinico-pathologic confirmed diagnosis, were included in the study. Results: A total of 1,628 scans were screened and initially 168 patients were identified. A subsequent total of 44 individuals (2.7% of the metastatic cohort) were included in this study, as having breast cancer with peritoneal secondaries. Of these, the majority (77%) had invasive ductal carcinomas (IDC). While the median survival from the diagnosis of metastatic breast cancer measured 20.5 months (range 0.1 -125 months), the median survival of patients with peritoneal disease was 1.56 months (range 0.2 - 27 months). Conclusions: Our data shows that the median survival of patients with peritoneal breast cancer metastasis is surprisingly poor, with only a minority surviving more than six months. A specific association with invasive lobular carcinoma (ILC) was not observed. The dismal outcome of these individuals despite further active therapy merits their inclusion into trials of new treatments. No significant financial relationships to disclose.