Abstract
The overall density of CD8+ tumor-infiltrating lymphocytes (TILs) is important for characterizing the level of immune activity in the tumor microenvironment (TME). Beyond the densities of ...CD8+ TILs, both their location and distributional patterns may also have relevance to immune activity. We evaluated 645 resected tumors encompassing seven cancer types, and correlate location and spatial patterns of CD8+ TILs to immune pathway activity.
We integrated image analysis results from digitized immunohistochemistry (IHC) slides with gene expression data from a targeted Ion Torrent Panel. Overall density of CD8+ TILs and the exact position of individual CD8+ lymphocytes were determined from IHC slides. A dissemination score was defined as ratio of global density and average local density of CD8+ TILs. This score is the inverse of the Ripley’s K statistic and becomes high for disseminated spatial patterns. We used this quotient as a continuous metric to identify tumors with a disseminated TIL pattern and to distinguish them from tumors with focal distribution of CD8+ TILs. Within a subset of tumors, the continuous dissemination metric was correlated with biological pathways using targeted mRNA sequencing and gene set enrichment analysis. In addition, association of the dissemination score with overall survival was tested on a subset of cases.
CD8+ TIL distributional patterns differed significantly between tumor types. Breast and pancreatic cancers more frequently showed a focal distribution of CD8+ TILs, while lung tumors comparatively exhibited a disseminated pattern. Transcriptional profiling data revealed differences between both image analysis phenotypes. On average, cases with more disseminated patterns of CD8+ T cells were associated with mRNA expression of genes that fall in pathways related to motility, migration and activation status of tumor infiltrating T cells. We also found a trend to better overall survival in patients whose tumors had a disseminated TIL score compared to those with a focal pattern. This trend was significant in non-small cell adenocarcinoma of the lung (log rank p = 0.018).
We demonstrate the value of spatial image analysis to automatically score CD8+ TIL dissemination as a marker of immune activity in the TME. Jointly analyzing transcriptional profiles appears to identify a biologically meaningful activation phenotype in tumors with high dissemination scores. Our data further suggests that this phenotype is associated with improved overall survival in some cancer patients.
Citation Format: Stefan Bentink, Andreas Spitzmueller, Tze Heng Tan, Hadassah Sade, Song Wu, Brandon W. Higgs, Keith E. Steele. Dissemination score of CD8+ TILs by automated image analysis is a potential marker of immune activity in human cancers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2795.
The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic ...infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.
Viral hemorrhagic fevers (VHFs) often cause high mortality with high infectivity, multiorgan failure, shock and hemorrhagic diathesis. Fibroblastic reticular cells (FRCs) within secondary lymphoid ...organs provide a supporting scaffold to T-lymphocyte areas. These cells regulate the movement of various immune cells and soluble molecules that promote T-lymphocyte homeostasis. We previously reported Ebola virus infection of FRCs, but ascribed little significance to this finding. Here, we studied infection of FRCs by Ebola, Marburg and Lassa viruses. We demonstrate that FRCs, or the extracellular 'conduit' of the fibroblastic reticulum of nonhuman primates, are targets of Ebola, Marburg and Lassa viruses. Furthermore, we observed that FRC damage correlates temporally and spatially with lymphocyte damage and that FRCs serve as nidi of fibrin deposition. In addition, we show that nonhuman primate FRCs express p75 NGF receptor and tissue transglutaminase. Our data suggest that viral infection of FRCs may be crucial to the immunological dysfunction and coagulopathy characteristic of VHFs. We further propose that p75 NGF receptor and tissue transglutaminase may be involved in FRC-associated dysfunction during the course of infection.
Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice ...necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls.
Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as ...many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring.
We developed a rat pilocarpine seizure/status epilepticus (SE) model, which closely resembles 1.6-2.0/LD50 soman exposure, to analyse the molecular mechanism of neuronal damage and to screen ...effective neuroprotectants against cholinergic agonist and chemical warfare nerve agent (CWNA) exposure. Rats implanted with radiotelemetry probes capable of recording electroencephalogram (EEG), electrocardiogram (ECG), temperature, and physical activity were treated with lithium chloride (5 mEq/kg, im), followed 24 h later by (ip) doses of pilocarpine hydrochloride. Based on radiotelemetry analysis, a dose of 240 mg/kg (ip) pilocarpine generated seizure/SE analogous to 1.6-2.0/LD50 of soman. The model was refined by reducing the peripheral convulsions without affecting the central nervous system (CNS) by administering methylscopolamine bromide (1 mg/kg, ip), an anti-cholinergic that does not cross the blood brain barrier. However, when methylscopolamine bromide was administered, a higher dose of pilocarpine (320 mg/kg, ip) was required to generate the equivalent seizure/SE. Histopathology data indicated that pilocarpine induces significant damage to the hippocampal region of the brain, with similar neuropathology to that of 1.6-2.0/LD50 soman exposure. There was a reduction in body temperature after the administration of pilocarpine, as observed in organophosphate (OP) nerve agents exposure. The heart-rate of pilocarpine-treated animals increased compared to the normal range. The pilocarpine seizure/SE model was also reproducible in the absence of lithium chloride. These results support that pilocarpine seizure/SE model is useful in studying the molecular mechanisms of neuropathology and screening neuroprotectants following cholinergic agonist and CWNA exposure.
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To evaluate stem cell-derived therapeutics for cutaneous vesicant injuries, we developed a dorsal exposure model using C57BL 6 black mice and half-mustard, 2-chloroethyl ethyl sulfide (CEES). The ...dorsal side of a mouse was exposed to 1-5 μl of CEES for 10 minutes and then decontaminated. The data demonstrate that 3 μl of CEES induced edema and erythema that peaked 24 h post exposure. Histopathology showed a central area of deep injury characterized by severe necrosis of epidermis and dermis. The C57BL 6 is a unique model that can be used to unravel the molecular mechanism of injury, identify the effects of black skin pigmentation, and evaluate the efficacy of stem cell therapeutics for cutaneous vesicant exposure.
For years the nation's development of medical countermeasures to biowarfare agents has primarily existed as the domain of the United States military, but it has taken on increased urgency in the last ...few years. The realization that the civilian population is also at risk from biological agents has resulted in the institution of new biodefense programs at a variety of nonmilitary organizations. USAMRIID, a long-time leader in the nation's biodefense effort, will soon be joined by other US government agencies as part of a planned National Interagency Biodefense Campus at Fort Detrick Maryland. US Army veterinary pathologists at USAMRIID have played an important role in the nation's biodefense effort, along with our veterinary colleagues representing other specialties, our military colleagues in other Army Medical Department corps, and our civilian colleagues. Together, we will continue to strive to develop the diagnostics, vaccines, therapeutic agents, and operational practices that are required to meet the great demands posed by the threat of biowarfare agents.
Stromal tumor-infiltrating lymphocytes (sTILs) are a potential predictive biomarker for immunotherapy response in metastatic triple-negative breast cancer (TNBC). To incorporate sTILs into clinical ...trials and diagnostics, reliable assessment is essential. In this review, we propose a new concept, namely the implementation of a risk-management framework that enables the use of sTILs as a stratification factor in clinical trials. We present the design of a biomarker risk-mitigation workflow that can be applied to any biomarker incorporation in clinical trials. We demonstrate the implementation of this concept using sTILs as an integral biomarker in a single-center phase II immunotherapy trial for metastatic TNBC (TONIC trial, NCT02499367), using this workflow to mitigate risks of suboptimal inclusion of sTILs in this specific trial. In this review, we demonstrate that a web-based scoring platform can mitigate potential risk factors when including sTILs in clinical trials, and we argue that this framework can be applied for any future biomarker-driven clinical trial setting.
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