bluuebunny/crossref_metadata_2025_split huggingface dataset:
7 Analytical comparison of a PD-L1 22C3 antibody laboratory-developed test protocol on the Benchmark XT and PD-L1 IHC 22C3 pharmDx: pan-tumor and triple-negative breast cancer samples
Authors: Vainer Gilad , Zatara Ghadeer , Huang Lingkang , Nuti Shanthy , Nuti Shanthy , Emancipator Kenneth 
Published: November 2021

BackgroundPD-L1 IHC 22C3 pharmDx is an FDA-approved companion diagnostic for pembrolizumab across multiple tumor types designed for use on the Autostainer Link 48 (AL48). Many pathology laboratories do not have access to the AL48 and therefore do not use PD-L1 IHC 22C3 pharmDx but instead assess PD-L1 using laboratory-developed tests (LDTs) on the Ventana BenchMark platform. We compared our PD-L1 22C3 antibody-based LDT on the BenchMark XT platform with PD-L1 IHC 22C3 pharmDx using cervical cancer (CC), head and neck squamous cell carcinoma (HNSCC), urothelial carcinoma (UC), esophageal SCC (ESCC), and triple-negative breast cancer (TNBC) samples.MethodsTumor specimens from patients with CC, HNSCC, UC, ESCC, and TNBC were stained with the 22C3 antibody, scored using the LDT on the BenchMark XT as previously described,1 and compared with PD-L1 IHC 22C3 pharmDx scored by a trained pathologist, who measured PD-L1 with the use of combined positive score (CPS) and standard cutoffs (HNSCC and CC, ≥1; UC, ESCC, and TNBC, ≥10). Agreement in PD-L1 CPS as determined using the LDT and the PD-L1 IHC 22C3 pharmDx was evaluated.Results423 samples with CC (n = 77), HNSCC (n = 126), UC (n = 121), ESCC (n = 80), and TNBC (n = 19) were evaluated in this study. The pan-tumor (CC, HNSCC, UC, and ESCC) intraclass correlation coefficient (ICC) of PD-L1 CPS as a continuous variable was 0.95 (95% CI, 0.94%–0.96%); Spearman correlations were 0.95. ICC (95% CI) was 0.92 (0.88–0.95) for CC, 0.97 (0.96–0.98) for HNSCC, 0.95 (0.92–0.97) for UC, and 0.92 (0.88–0.95) for ESCC; Spearman correlation was 0.93, 0.96, 0.92, and 0.89, respectively. The overall percentage agreement at the respective CPS cutoff was 96% (CC), 96% (HNSCC), 96% (UC), and 90% (ESCC). Staining patterns by 22C3 LDT and PD-L1 IHC 22C3 pharmDx were also very similar in our TNBC pilot study; however, correlation was not calculated because of the small sample numbers.ConclusionsThe PD-L1 22C3 antibody-based LDT on the BenchMark XT demonstrated high concordance with PD-L1 IHC 22C3 pharmDx. These findings suggest the comparability of PD-L1 IHC 22C3 pharmDx with an LDT based on the 22C3 antibody across several tumor types. Further validation for TNBC is ongoing to confirm the data from the pilot run.ReferenceNeuman T, et al. J Thorac Oncol. 2016;11:1863–1868.
8 Multiparameter characterization of CAR T cells
Authors: Wang Xueting , Pitzka Christina , Rheindorf Daniela , Mockel-Tenbrinck Nadine , Holzer Tatjana , Richter Anne , Cathomen Toni , Evaristo Cesar 
Published: November 2021

BackgroundAdoptive cell transfer of chimeric antigen receptor (CAR) modified T cells has demonstrated great therapeutic success against certain hematological malignancies. However, a substantial number of patients experienced relapse at some point after treatment with the underlying mechanisms not fully understood. Emerging data suggest that the undesired clinical outcome is related to different aspects, which include: the tumor heterogeneity, the tumor microenvironment, as well as intrinsic characteristics of the CAR T cells. In this work, we aimed to understand the diversity of CAR T cells generated from different donors, using multiparameter in vitro characterization.MethodsLeukapheresis from healthy donors were collected to generate CAR T cells using the GMP-compliant CliniMACS Prodigy® platform, enabling an automated and closed engineering of CAR T cells in a highly reproducible manner. We performed an in-depth characterization of the resulting CAR T cells by exploring differences in the immunophenotype, cell fitness and effector function of the freshly prepared as compared to frozen CAR T cell samples. Specifically, we designed several flow cytometry panels for the extensive characterization of immunophenotypes of interest such as: proliferative capacity, differentiation, activation and exhaustion. Cell fitness status was determined by the rate at which cells undergo apoptosis following stress. Finally, effector function was determined by the ability of the activated CAR T cells to secrete proinflammatory cytokines including IFN-g, TNF-a and IL-2. The associations between these different parameters were analyzed using comprehensive statistical approaches.ResultsWith our established workflow, over 20 healthy-donor derived CAR T cells were generated and characterized. We have observed donor-dependent variations and responses for most of the explored parameters. In general, the freezing and thawing process negatively affected cell fitness and effector function of the CAR T cells and resulted in altered immunophenotypes. Additionally, correlations between certain immunophenotypes and cell fitness/effector function were identified.ConclusionsCollectively, we established a workflow for multiparameter characterization of CAR T cells and assessed the intrinsic variability of CAR T cells for both research and clinical application.
29 Elevated Flt3L predicts long-term survival in patients with high-grade gastroenteropancreatic neuroendocrine neoplasms
Authors: Detjen Katharina , Otto Raik , Giesecke Yvonne , Geisler Lukas , Riemer Pamela , Jann Henning , Grötzinger Carsten , Sers Christine , Luedde Tom , Leser Ulf , Wiedenmann Bertram , Sigal Michael , Tacke Frank , Roderburg Christoph , Hammerich Linda 
Published: November 2021

BackgroundGastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) are a rare and heterogeneous family of tumors arising from the disseminated neuroendocrine system of the gastrointestinal tract and pancreas. Clinical management of high-grade GEP-NEN is challenging due to disease heterogeneity, illustrating the need for reliable biomarkers facilitating patient stratification and guiding treatment decisions. FMS-like tyrosine kinase 3 ligand (Flt3L) is emerging as a prognostic or predictive surrogate marker of host tumoral immune response and might enable stratification of patients with otherwise comparable tumor features.MethodsWe used RNAseq data from human foregut-derived pancreatic and gastric GEP-NEN to evaluate Flt3L gene expression in tumor tissue. The data set (n=54) represented the full range of NEN grades and differentiation, and expression levels were compared to healthy control tissue as well. We also analyzed circulating Flt3L levels in serum samples of a separate cohort of G2/G3 GEP-NEN (n=59) an healthy controls (n=4). The study was approved by the local ethics committee at Charité Universitätsmedizin Berlin, Germany (ethical approval number EA1/229/17) and patient informed consent was obtained.ResultsWe detected a prominent induction of Flt3L gene expression in individual G2 and G3 NEN, but not in G1 neuroendocrine tumors (NET). Flt3L mRNA expression levels in tumor tissue predicted disease related survival of patients with highly proliferative G2 and G3 NEN more accurately than the conventional criteria of grading or NEC/NET differentiation. High level Flt3L mRNA expression was associated with increased expression of genes related to immunogenic cell death, lymphocyte effector function and dendritic cell maturation, suggesting a less tolerogenic (more proinflammatory) phenotype of tumors with Flt3L induction. Importantly, circulating levels of Flt3L were also elevated in high grade NEN and correlated with patients´ progression-free and disease-related survival, thereby reflecting the results observed in tumor tissue.ConclusionsOur results suggest Flt3L as a surrogate marker of an inflammatory tumor microenvironment. Therefore, we propose Flt3L as a prognostic biomarker for high grade GEP-NEN. Flt3L measurements in serum, which can be easily be incorporated into clinical routine, may hold the promise to guide patient stratification and tailor treatment decisions and should be further evaluated, especially in the context of immunotherapies.Ethics ApprovalThe study was approved by the local ethics committee at Charité Universitätsmedizin Berlin, Germany (ethical approval number EA1/229/17) and patient informed consent was obtained.
50 In-situ visualization and measurement of tumor-infiltrating lymphocytes (TILs) on intact FFPE renal cell carcinoma (RCC) tissue using the spatial molecular imager (SMI)
Authors: Newell Evan , Kim Youngmi , Ryu Heeju , Li Shamin , Leon Michael , Kim Sean , Gregory Mark , Danaher Patrick , Beechem Joseph 
Published: November 2021

BackgroundAlthough cancer immunotherapies can effectively restore T cell-mediated immunity leading to sustained clinical responses, these responses are unpredictable partly due to highly heterogeneous phenotypes of tumor-infiltrating lymphocytes (TILs) between patients. Thus, understanding such TILs and their roles in the context of tumor microenvironments (TME) may lead to developing better immunotherapy solutions. The spatial molecular imager (SMI) is a novel spatial transcriptomics platform that allows spatially resolved high-dimensional cellular phenotyping for comprehensive TIL profiling. SMI uses fluorescent molecular barcodes to enable in-situ measurement of biological targets on an intact tissue sample. Here, we characterize comprehensive TIL phenotypes and visualize landscape of TILs directly on intact formalin-fixed paraffin-embedded (FFPE) tissues using a 1000+-plex RNA panel.MethodsTo build multi-omics TIL profiling data sets for renal cell carcinoma (RCC) tissues, we employed scRNA-seq, mass cytometry (CyTOF) and SMI. Peripheral blood mononuclear cells and dissociated cells from matched RCC tumor and adjacent normal tissues were analyzed by CyTOF and single-cell sequencing. Then, SMI profiling of matching FFPE tissues was used to visualize TILs in the context of the TME and to understand relationships between high-dimensional cellular heterogeneity and the spatial organization of cells within a tumor tissue.ResultsCyTOF and scRNA-seq analysis of dissociated cells was used to determine the gene expression profiles of numerous cellular subsets. TCR sequencing was also used to assess the extent of clonal expansion and clonotypic relationships between blood and tumor. Consistent with our previous reports, T cell populations could be segregated based on markers associated with chronic T cell receptor signaling and many T cells with an exhausted phenotype were clonally expanded in the tumor but not the blood. In contrast, T cell clonotypes with bystander phenotypes in the tumor were readily detected as expanded clones in the blood, supporting notion that not all tumor-infiltrating T cells are specific for tumor antigens. SMI analysis of matched tumor tissue was used to accurately quantify the densities and to determine the spatial organization of all T cell subsets. In addition, computational methods were used to describe distinct cellular niches within tumors with accurately defined cellular compositions.ConclusionsHigh dimensional cellular profiling highlights the abundance of bystander T cell infiltration of RCC tumors. Comprehensive spatial profiling by SMI provides spatial context to the highly diverse immune cell composition of tumor infiltrates.Ethics ApprovalFully anonymous human material was obtained from Northwest Biotrust and given IRB designation of non-human subjects research.
75 Comprehensive profiling of the tumor-immune microenvironment using an augmented transcriptome
Authors: Levy Eric , Milani Pamela , Navarro Fabio , Bartha Gabor , Abbott Charles , Jacob Jose , McClory Rena , Li Robin , West John , Lyle John , Boyle Sean , Chen Richard 
Published: November 2021

BackgroundComprehensive profiling of both the tumor and tumor microenvironment (TME) can help further our understanding of tumor progression and response to treatment. Many immune features can be extracted from transcriptomic data, including characterization of the immune infiltrate and profiling the diversity of immune receptors. To address this, we have developed multiple TME profiling features as part of the ImmunoID NeXT Platform®, an augmented, immuno-oncology-optimized exome/transcriptome platform designed to provide comprehensive information regarding the tumor and TME from a single FFPE tumor sample. These features including quantification of immune cell infiltration and profiling of the T-cell receptor (TCR) and B-cell receptor (BCR).MethodsTo develop our immune infiltrate quantification method, we profiled the transcriptomes of eight purified immune cell types using ImmunoID NeXT™ to develop platform-specific gene sets, and compared our transcriptome quantification to immune cell quantification with IHC. For TCR and BCR methods, we analyzed the reproducibility of clone results, and compared top clones to standalone TCR and BCR sequencing approaches. In addition, we characterized the immune content of over 800 tumor samples across 14 cancer types. Finally, we analyzed the immune features in a cohort of melanoma patients who underwent PD-1 blockade.ResultsWe observe significant concordances between cell fractions by IHC and ImmunoID NeXT’s transcriptome-based scores in tumor FFPE samples for B cells, CD8+ T cells, and macrophages (R2>0.82, R2>0.75, and R2>0.52, respectively). For TCR and BCR methods, abundances of clones shared between subsequent curls of a tumor FFPE sample have very high concordances (R2>0.89, R2>0.92, and R2>0.76 for TRB, IgG, and IgA, respectively). Compared to the standalone approaches, we identify 100% of the top 500 TRB clones and 95% of the top 500 IgG clones, with highly concordant abundances (R2>0.94 and R2>0.82 for TRB and IgG, respectively) in a PBMC sample. We identify biologically-relevant immune signatures across tumor types by characterizing the immune features across over 800 tumor samples. Finally, in a melanoma cohort, TRB clonality and CD8+ T cell scores are significantly different in responders to checkpoint inhibition.ConclusionsRNA sequencing can be used as a scalable approach to profile the immune composition in tumors. Such analysis can add to our understanding of the tumor-immune interaction, including studies of response to immunotherapy. We show that immune infiltrate quantification and TCR and BCR profiling – all part of the ImmunoID NeXT Platform – are able to accurately and effectively evaluate the composition and diversity of tumor-infiltrating immune cells.
92 Single cell and spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the novel RNAscope™ HiPlex v2 in situ hybridization assay
Authors: Basak Sayantani , Dikshit Anushka , Yu Ming , Ji HaYeun , Chang Ching-Wei , Zhang Bingqing 
Published: November 2021

BackgroundThe tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. Understanding spatial interactions between various cell types and their activation states in the TME is crucial for implementing successful immunotherapy strategies against various types of cancer. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in human lung, breast, cervical and ovarian FFPE tumor tissues.MethodsWe have expanded our current RNAscope HiPlex assay capability of iteratively multiplexing up to 12 targets in fixed and fresh frozen samples to include formalin fixed paraffin embedded (FFPE) tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. We have leveraged this technology to investigate spatial expression of 12 oncology and immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokine RNA expression profile within the TME. The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction.ResultsWe visualized T cell infiltration and identified T cell subsets within tumors using CD3and CD8 expression and activated T cells by IFNG expression. We further identified subsets of pro- and anti-inflammatory macrophages by CD68 and CD163 expression as well effector cells which secrete chemokines and cytokine. We also detected the hypoxia markers HIF1A and VEGF to elucidate the immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis software showed higher copy numbers in the lung tumor as compared to the other tumors, demonstrating the sensitivity of the assay through differential expression. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME.ConclusionsUsing a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, we have demonstrated the capability of this technique to spatially resolve 12 targets in four different tumor types. The FFPE reagent efficiently quenched background autofluorescence in the tissues and identified immune cell signatures within the TME. Quantification of immunosuppressive markers further depicted a differential expression among various tumors. This technology is highly beneficial for investigating complex and spatial tumor-stroma interactions in basic science and translational research. The assay can also provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues.
306 Decr2 regulates tumor cell ferroptosis and is required for immunotherapy efficacy
Authors: Li Shuyin , Gajewski Thomas , Li Shuyin , Higgs Emily 
Published: November 2021

BackgroundCheckpoint blockade therapies have transformed the landscape of cancer care. Durable clinical responses have been observed in a subset of patients. However, many patients do not respond, and understanding the mechanisms underlying tumor resistance to checkpoint blockade drugs could potentially expand clinical benefit. We therefore performed an unbiased CRISPR library screen to identify novel gene alterations sufficient to mediate resistance to T cell-mediated killing.MethodsB16.SIY cells were transduced with a genome-scale gRNA lentivirus to generate loss of function mutants. In vitro-primed CD8+ T cells isolated from 2C/Rag2–/– TCR transgenic mice specific for the SIY antigen were co-cultured with transduced B16.SIY tumor cells. Resistant cells were collected and targeted genes were identified by sequencing the gRNAs. The gene encoding Decr2, a peroxisomal 2,4-dienoyl-CoA reductase, was identified. To investigate the role of Decr2 in tumor growth and immune responses in vivo, the Decr2 knock-down or Decr2 overexpressed tumors were transplanted into C57BL/6 mice for studies of anti-PD-L1 efficacy and immune response analysis. Based on a hypothesized role in ferroptosis, RNA-seq was performed and lipid reactive oxygen species were quantified.ResultsDecr2 mutants were relatively resistant to CD8+ T cell killing in vitro. In vivo, Decr2 knock-down tumors showed almost complete loss of anti-PD-L1 efficacy, without affecting early T cell priming or immune cell infiltration. However, tumor-infiltrating T cells failed to expand following anti-PD-L1 therapy in Decr2 knock-down tumors. RNA-seq analysis revealed upregulation of ferroptosis-related genes in Decr2-deficient tumor cells, and these tumor cells were relatively resistant to ferroptosis-inducers in vitro. Decr2 knock-down cells also showed defective induction of lipid ROS in response to CD8+ T cells in vitro, or when analyzed following anti-PD-L1 treatment ex vivo. Analysis of tumor RNA-seq data from human melanoma patients treated with checkpoint blockade revealed decreased Decr2 levels in non-responding patients.ConclusionsOur results identify tumor cell-expressed Decr2 as novel regulator of ferroptosis and implicate the process of ferroptosis as a major mechanism of immune-mediated tumor cell killing in vivo. Pharmacologic strategies to facilitate tumor cell ferroptosis could have therapeutic utility.
Polar bear (Ursus maritimus) use of the Cape Bathurst polynya and flaw lead
Authors: Henderson Erin M. , Derocher Andrew E. 
Published: November 2021

The Cape Bathurst polynya and flaw lead (CBP) are major, predictable habitat features with ≤15% ice cover in an otherwise ice-covered Beaufort Sea, and thought to provide hunting opportunities for polar bears (Ursus maritimus Phipps, 1774). We assessed 78 adult (female; with and without cubs) and subadult (male and female) polar bears’ use of the CBP from October to June 2007–2014. The CBP was up to 725 km wide in autumn, ice-covered in winter, and <306 km wide in spring. Seventy-nine percent (n = 62) of the bears used the CBP (≥1 location <2.4 km, or one 4 h step length, from the CBP). Use was higher for solitary adult females and subadult males, which travelled faster with low turning angles along wider sections than females with offspring and subadult females. Bears were closest to the CBP during the spring hyperphagia season. Although a wider CBP did not prevent crossing, bears primarily crossed from the coast towards pack ice at locations 53% narrower than areas not crossed. Bears might avoid crossing when it would require a long-distance swim. The CBP affects polar bear ecology by providing hunting habitat and a corridor that could increase prey encounters but may affect movement.
Prolonged SARS-CoV-2 Infection in Patients with Lymphoid Malignancies
Authors: Lee Christina Y. , Shah Monika K. , Hoyos David , Solovyov Alexander , Douglas Melanie , Taur Ying , Maslak Peter , Babady N. Esther , Greenbaum Benjamin , Kamboj Mini , Vardhana Santosha A. 
Published: November 2021

Abstract

Coronavirus disease 2019 (COVID-19) infection results in both acute mortality and persistent and/or recurrent disease in patients with hematologic malignancies, but the drivers of persistent infection in this population are unknown. We found that B-cell lymphomas were at particularly high risk for persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positivity. Further analysis of these patients identified discrete risk factors for initial disease severity compared with disease chronicity. Active therapy and diminished T-cell counts were drivers of acute mortality in COVID-19–infected patients with lymphoma. Conversely, B cell–depleting therapy was the primary driver of rehospitalization for COVID-19. In patients with persistent SARS-CoV-2 positivity, we observed high levels of viral entropy consistent with intrahost viral evolution, particularly in patients with impaired CD8+ T-cell immunity. These results suggest that persistent COVID-19 infection is likely to remain a risk in patients with impaired adaptive immunity and that additional therapeutic strategies are needed to enable viral clearance in this high-risk population.

Significance:
We describe the largest cohort of persistent symptomatic COVID-19 infection in patients with lymphoid malignancies and identify B-cell depletion as the key immunologic driver of persistent infection. Furthermore, we demonstrate ongoing intrahost viral evolution in patients with persistent COVID-19 infection, particularly in patients with impaired CD8+ T-cell immunity.
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