Journal Articles

Innovative Methods

Methods with focus on improving cancer immunotherapy approaches

T cell responses in the microenvironment of primary renal cell carcinoma - Implications for adoptive cell therapy

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In vitro expansion of large numbers of highly potent tumor-reactive T cells appears a prerequisite for effective adoptive cell therapy (ACT) with autologous tumor-infiltrating lymphocytes (TIL) as shown in metastatic melanoma (MM). We therefore sought to determine whether renal cell carcinomas (RCC) are infiltrated with tumor-reactive T cells that could be efficiently employed for adoptive transfer immunotherapy. TILs and autologous tumor cell lines (TCLs) were successfully generated from 22 (92%) and 17 (77%) of 24 consecutive primary RCC specimens and compared to those generated from MM. Immune recognition of autologous TCLs or fresh tumor digests (FTD) was observed in CD8+ TILs from 82% of patients (18/22). Cytotoxicity assays confirmed the tumoricidal capacity of RCC-TILs. The overall expansion capacity of RCC-TILs was similar to MM-TILs. However, the magnitude, poly-functionality, and ability to expand in classical expansion protocols of CD8+ T-cell responses was lower compared to MM-TILs. The RCC-TILs that did react to the tumor were functional and antigen presentation and processing on RCC-tumors was similar to MM-TILs. Direct recognition of tumors with cytokine-induced overexpression of human leukocyte antigen (HLA) class II was observed from CD4+ T cells (6/12; 50%). Thus, TILs from primary RCC specimens could be isolated, expanded, and could recognize tumors. However, immune responses of expanded CD8+ RCC-TILs were typically weaker than MM-TILs and displayed a mono-/oligo- functional pattern. The ability to select, enrich, and expand tumor-reactive poly-functional T cells may be critical in developing effective ACT with TILs for RCC.

Author Info: (1) Department of Hematology, Center for Cancer Immune Therapy, Herlev Hospital, University of Copenhagen. (2) Department of Hematology, Center for Cancer Immune Therapy, Herlev Hospital

Author Info: (1) Department of Hematology, Center for Cancer Immune Therapy, Herlev Hospital, University of Copenhagen. (2) Department of Hematology, Center for Cancer Immune Therapy, Herlev Hospital, University of Copenhagen. (3) Department of Hematology, Center for Cancer Immune Therapy, Herlev Hospital, University of Copenhagen. (4) Institute of Medical Immunology, Martin Luther University Halle-Wittenberg. (5) Division for Immunology and Vaccinology, Technical University of Denmark. (6) Division for Immunology and Vaccinology, Technical University of Denmark. (7) Department of Oncology, Herlev Hospital, University of Copenhagen. (8) Institute of Medical Immunology, Martin Luther University Halle-Wittenberg. (9) Department of Urology, Herlev Hospital, University of Copenhagen. (10) Department of Pathology, Herlev Hospital, University of Copenhagen. (11) Department of Hematology, Center for Cancer Immune Therapy, Herlev Hospital, University of Copenhagen. (12) Department of Hematology, Center for Cancer Immune Therapy, Herlev Hospital, University of Copenhagen inge.marie.svane@regionh.dk.

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Reagent Tracker Dyes Permit Quality Control for Verifying Plating Accuracy in ELISPOT Tests

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ELISPOT assays enable the detection of the frequency of antigen-specific T cells in the blood by measuring the secretion of cytokines, or combinations of cytokines, in response to antigenic challenges of a defined population of PBMC. As such, these assays are suited to establish the magnitude and quality of T cell immunity in infectious, allergic, autoimmune and transplant settings, as well as for measurements of anti-tumor immunity. The simplicity, robustness, cost-effectiveness and scalability of ELISPOT renders it suitable for regulated immune monitoring. In response to the regulatory requirements of clinical and pre-clinical immune monitoring trials, tamper-proof audit trails have been introduced to all steps of ELISPOT analysis: from capturing the raw images of assay wells and counting of spots, to all subsequent quality control steps involved in count verification. A major shortcoming of ELISPOT and other related cellular assays is presently the lack of audit trails for the wet laboratory part of the assay, in particular, the assurance that no pipetting errors have occurred during the plating of antigens and cells. Here, we introduce a dye-based reagent tracking platform that fills this gap, thereby increasing the transparency and documentation of ELISPOT test results.

Author Info: (1) Research and Development Department, CTL, Shaker Heights, OH 44122, USA. alexander.lehmann@immunospot.com. (2) Research and Development Department, CTL, Shaker Heights, OH 44122, USA. zoltan.megyesi@immunospot.com. (3)

Author Info: (1) Research and Development Department, CTL, Shaker Heights, OH 44122, USA. alexander.lehmann@immunospot.com. (2) Research and Development Department, CTL, Shaker Heights, OH 44122, USA. zoltan.megyesi@immunospot.com. (3) Research and Development Department, CTL, Shaker Heights, OH 44122, USA. anna.przybyla@immunospot.com. Department of Cancer Immunology, Chair of Medical Biotechnology, Poznan University of Medical Sciences, 61-701 Poznan, Poland. anna.przybyla@immunospot.com. (4) Research and Development Department, CTL, Shaker Heights, OH 44122, USA. paul.lehmann@immunospot.com.

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Antigen-specific antitumor responses induced by OX40 agonist are enhanced by IDO inhibitor indoximod

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Although an immune response to tumors may be generated using vaccines, so far, this approach has only shown minimal clinical success. This is attributed to the tendency of cancer to escape immune surveillance via multiple immune suppressive mechanisms. Successful cancer immunotherapy requires targeting these inhibitory mechanisms along with enhancement of antigen-specific immune responses to promote sustained tumor-specific immunity. Here we evaluated the effect of indoximod, an inhibitor of the immunosuppressive indoleamine-(2,3)-dioxygenase (IDO) pathway, on antitumor efficacy of anti-OX40 agonist in the context of vaccine in the IDO- TC-1 tumor model. We demonstrate that although the addition of anti-OX40 to the vaccine moderately enhances therapeutic efficacy, incorporation of indoximod into this treatment leads to enhanced tumor regression and cure of established tumors in 60% of treated mice. We show that the mechanisms by which the IDO inhibitor leads to this therapeutic potency include (i) an increment of vaccine-induced tumor-infiltrating effector T cells that is facilitated by anti-OX40, and (ii) a decrease of IDO enzyme activity produced by non-tumor cells within the tumor microenvironment that results in enhancement of the specificity and the functionality of vaccine-induced effector T cells. Our findings suggest a translatable strategy to enhance the overall efficacy of cancer immunotherapy.

Author Info: (1) Georgia Cancer Center, Augusta University. (2) Georgia Cancer Center, Augusta University. (3) Georgia Cancer Center, Augusta University. (4) Georgia Cancer Center, Augusta University. (5)

Author Info: (1) Georgia Cancer Center, Augusta University. (2) Georgia Cancer Center, Augusta University. (3) Georgia Cancer Center, Augusta University. (4) Georgia Cancer Center, Augusta University. (5) Georgia Cancer Center, Augusta University. (6) Georgia Cancer Center, Augusta University. (7) Georgia Cancer Center, Augusta University. (8) Georgia Cancer Center, Augusta University. (9) The University of Aberdeen Dental School & Hospital, The Institute of Medicine, Medical Sciences & Nutrition, The University of Aberdeen. (10) Medimmune Inc. (11) Georgia Cancer Center, Augusta University. (12) Georgia Cancer Center, Augusta University skhleif@augusta.edu.

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Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer

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Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime are required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-gamma cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

Author Info: (1) Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal; Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; Center for

Author Info: (1) Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal; Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Faculty of Medicine (Polo I), Coimbra, Portugal. (2) Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. (3) Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. (4) Department of Physiology and Pharmacology, Sackler School of Medicine, Room 607, Tel Aviv University, Tel Aviv, Israel. (5) Flow Cytometry unit, Biological Services Department, Weizmann Institute of Science, Rehovot, Israel. (6) Chemistry and Biochemistry Center, Sciences Faculty, Universidade de Lisboa, Lisbon, Portugal. (7) Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; Immunology research center, Tel Aviv Sourasky Medical Center (TASMC), Tel Aviv, Israel. (8) Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; Immunology research center, Tel Aviv Sourasky Medical Center (TASMC), Tel Aviv, Israel. (9) Department of Immunology, Weizmann Institute of Science, Rehovot, Israel; Immunology research center, Tel Aviv Sourasky Medical Center (TASMC), Tel Aviv, Israel. (10) Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Faculty of Medicine (Polo I), Coimbra, Portugal;; Faculty of Pharmacy (FFUC), University of Coimbra, Polo das Ciencias da Saude, Azinhaga de Santa Comba, Coimbra, Portugal. (11) Department of Physiology and Pharmacology, Sackler School of Medicine, Room 607, Tel Aviv University, Tel Aviv, Israel. (12) Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. (13) Department of Immunology, Weizmann Institute of Science, Rehovot, Israel. (14) Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal. Electronic address: hflorindo@ff.ul.pt.

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Supramolecular Peptide Nanofibers Engage Mechanisms of Autophagy in Antigen-Presenting Cells

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Supramolecular peptide nanofibers are attractive for applications in vaccine development due to their ability to induce strong immune responses without added adjuvants or associated inflammation. Here, we report that self-assembling peptide nanofibers bearing CD4+ or CD8+ T cell epitopes are processed through mechanisms of autophagy in antigen-presenting cells (APCs). Using standard in vitro antigen presentation assays, we confirmed loss and gain of the adjuvant function using pharmacological modulators of autophagy and APCs deficient in multiple autophagy proteins. The incorporation of microtubule-associated protein 1A/1B-light chain-3 (LC3-II) into the autophagosomal membrane, a key biological marker for autophagy, was confirmed using microscopy. Our findings indicate that autophagy in APCs plays an essential role in the mechanism of adjuvant action of supramolecular peptide nanofibers.

Author Info: (1) Department of Pharmacology & Toxicology, Department of Microbiology and Immunology, and Sealy Center for Vaccine Development, University of Texas Medical Branch, 301 University Blvd

Author Info: (1) Department of Pharmacology & Toxicology, Department of Microbiology and Immunology, and Sealy Center for Vaccine Development, University of Texas Medical Branch, 301 University Blvd, Route 0617, Galveston, Texas 77555, United States. Department of Pharmacology & Toxicology, Department of Microbiology and Immunology, and Sealy Center for Vaccine Development, University of Texas Medical Branch, 301 University Blvd, Route 0617, Galveston, Texas 77555, United States. (2) Immunobiology and Transplant Science Center, Houston Methodist Research Institute, 6565 Fannin Street, Houston, Texas 77030, United States. (3) Department of Pharmacology & Toxicology, Department of Microbiology and Immunology, and Sealy Center for Vaccine Development, University of Texas Medical Branch, 301 University Blvd, Route 0617, Galveston, Texas 77555, United States. (4) Department of Pharmacology & Toxicology, Department of Microbiology and Immunology, and Sealy Center for Vaccine Development, University of Texas Medical Branch, 301 University Blvd, Route 0617, Galveston, Texas 77555, United States. Department of Pharmacology & Toxicology, Department of Microbiology and Immunology, and Sealy Center for Vaccine Development, University of Texas Medical Branch, 301 University Blvd, Route 0617, Galveston, Texas 77555, United States. (5) Division of Surgical Oncology, Robert Wood Johnson Medical School, Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, RM 3035, New Brunswick, New Jersey 08903, United States. (6) Immunobiology and Transplant Science Center, Houston Methodist Research Institute, 6565 Fannin Street, Houston, Texas 77030, United States. (7) Department of Pathology and Laboratory Medicine, McGovern Medical School, University of Texas Health Science Center at Houston, 6431 Fannin Street, P.O. Box 20708, Houston, Texas 77030, United States.

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The reproducibility of PD-L1 scoring in lung cancer: can the pathologists do better

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In the era of personalized medicine, the selection of advanced stages non-small cell lung cancer (NSCLC) patients for targeted treatments requires development, validation and continuous quality assessments of a wide array of laboratory assays, including both conventional and developing methodologies. While high throughput molecular testing approaches, to extensively assess genomic biomarkers of current and potential clinical value, are fascinating innovations in the field of modern oncology, traditional morpho-molecular methodologies such as fluorescent in situ hybridisation and immunohistochemical (IHC) techniques are still precious in the clinic to guide therapeutic interventions (1). This holds even more true, when considering the recent requirements to evaluate in NSCLC cells the checkpoint inhibitor programmed cell death ligand 1 (PD-L1) protein expression. Different primary antibody clones, raised against different epitopes (parts) of the PD-L1, are available (2). Each clone is linked to a specific treatment: clone 28-8 (Dako, Glostrup, Denmark) for nivolumab, 22C3 (Dako) for pembrolizumab, SP142 (Ventana, Tucson, AZ, USA) for atezolizumab and SP263 (Ventana) for durvalumab. Different clinical trial performed its own PD-L1 immunohistochemistry assay as a prepackaged kit of reagents running on company-specific staining platforms according to the manufacturersinstructions either on the Dako Link AS-48 (no longer available commercially) or on the Ventana Benchmark autostainer systems, adopting custom scoring-criteria for each assay (2)

Author Info: (1) Department of Public Health, University of Naples Federico II, Naples, Italy. (2) Division of Medical Oncology, "S.G. Moscati" Hospital, Avellino, Italy.

Author Info: (1) Department of Public Health, University of Naples Federico II, Naples, Italy. (2) Division of Medical Oncology, "S.G. Moscati" Hospital, Avellino, Italy.

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Study of the tumor microenvironment during breast cancer progression

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Background: Different cells and mediators in the tumor microenvironment play important roles in the progression of breast cancer. The aim of this study was to determine the composition of the microenvironment during tumor progression in order to discover new related biomarkers and potentials for targeted therapy. Methods: In this study, breast cancer biopsies from four different stages, and control breast biopsies were collected. Then, the mRNA expression of several markers related to different CD4(+) T cell subsets including regulatory T cells (Treg), T helper (Th) type 1, 2 and 17 were determined. In addition, we investigated the expression of two inflammatory cytokines (TNF-alpha and IL-6) and inflammatory mediators including FASL, IDO, SOCS1, VEGF, and CCR7. Results: The results showed that the expression of Th1 and Th17 genes was decreased in tumor tissues compared to control tissues. In addition, we found that the gene expression related to these two cell subsets decreased during cancer progression. Moreover, the expression level of TNF-alpha increased with tumor progression. Conclusion: We conclude that the expression of genes related to immune response and inflammation is different between tumor tissues and control tissues. In addition, this difference was perpetuated through the different stages of cancer.

Author Info: (1) Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.0000 0001 0166 0922grid.411705.6 (2) Genetics Department, Breast Cancer Research Center, Motamed

Author Info: (1) Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.0000 0001 0166 0922grid.411705.6 (2) Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran.grid.417689.5 (3) Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.0000 0001 0166 0922grid.411705.6 (4) Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran.0000 0001 0166 0922grid.411705.6 (5) Inflammation Research Network-Snyder Institute for Chronic Disease, Department of Physiology and Pharmacology, University of Calgary Cumming School of Medicine, Calgary, AB Canada.0000 0004 1936 7697grid.22072.35 (6) Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.0000 0001 1781 3962grid.412266.5 (7) Medical Plants Research Center, Shahrekord University of Medical Sciences, Shahrekord, Iran.0000 0004 0384 8883grid.440801.9 (8) Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.0000 0001 0166 0922grid.411705.6 (9) Genetics Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran.grid.417689.5

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Translating Science into Survival: Report on the Third International Cancer Immunotherapy Conference

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On September 6 to 9, 2017, in Mainz, Germany, the Third International Cancer Immunotherapy Conference was hosted jointly by the Cancer Research Institute, the Association for Cancer Immunotherapy, the European Academy of Tumor Immunology, and the American Association for Cancer Research. For the third straight year, more than 1,400 people attended the four-day event, which covered the latest advances in cancer immunology and immunotherapy. This report provides an overview of the main topics discussed. Cancer Immunol Res; 6(1); 10-13. (c)2017 AACR.

Author Info: (1) TRON - Translational Oncology, University Medical Center of Johannes Gutenberg University, Mainz, Germany. (2) TRON - Translational Oncology, University Medical Center of Johannes Gutenberg

Author Info: (1) TRON - Translational Oncology, University Medical Center of Johannes Gutenberg University, Mainz, Germany. (2) TRON - Translational Oncology, University Medical Center of Johannes Gutenberg University, Mainz, Germany. (3) Cancer Research Institute, New York, New York. abrodsky@cancerresearch.org.

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Immunomodulatory effects of soluble CD5 on experimental tumor models

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Modulation of antitumor immune responses by targeting immune checkpoint regulators has been proven successful in the treatment of many different tumors. Recent evidence shows that the lymphocyte receptor CD5 -a negative regulator of TCR-mediated signaling- may play a role in the anti-tumor immune response. To explore such an issue, we developed transgenic C57BL/6 mice expressing a soluble form of human CD5 (shCD5EmuTg), putatively blocking CD5-mediated interactions ("decoy receptor" effect). Homozygous shCD5EmuTg mice showed reduced growth rates of tumor cells of melanoma (B16-F0) and thymoma (EG7-OVA) origin. Concomitantly, increased CD4(+) and CD8(+) T cell numbers, as well as reduced proportion of CD4(+)CD25(+)FoxP3(+) (Treg) cells were observed in tumor draining lymph nodes (TdLN). TdLN cell suspensions from tumor-bearing shCD5EmuTg mice showed increased both tumor specific and non-specific cytolitic activity. Moreover, subcutaneous peritumoral (p.t.) injection of recombinant shCD5 to wild-type (WT) mice slowed B16-F0 tumor growth, and reproduced the above mentioned TdLN cellular changes. Interestingly, lower intratumoral IL-6 levels -an inhibitor of Natural Killer (NK) cell cytotoxity- were observed in both transgenic and rshCD5-treated WT mice and the anti-tumor effect was abrogated by mAb-induced NK cell depletion. Taken together, the results further illustrate the putative regulatory role of CD5-mediated interactions in anti-tumor immune responses, which would be at least in part fostered by NK cells.

Author Info: (1) Immunoreceptors of the Innate and Adaptive System, Institut d'Investigacions Biomediques August Pi i Sunyer, 08036, Barcelona, Spain. (2) Immunoreceptors of the Innate and Adaptive

Author Info: (1) Immunoreceptors of the Innate and Adaptive System, Institut d'Investigacions Biomediques August Pi i Sunyer, 08036, Barcelona, Spain. (2) Immunoreceptors of the Innate and Adaptive System, Institut d'Investigacions Biomediques August Pi i Sunyer, 08036, Barcelona, Spain. (3) Immunoreceptors of the Innate and Adaptive System, Institut d'Investigacions Biomediques August Pi i Sunyer, 08036, Barcelona, Spain. (4) Immunoreceptors of the Innate and Adaptive System, Institut d'Investigacions Biomediques August Pi i Sunyer, 08036, Barcelona, Spain. (5) Immunoreceptors of the Innate and Adaptive System, Institut d'Investigacions Biomediques August Pi i Sunyer, 08036, Barcelona, Spain. (6) Immunoreceptors of the Innate and Adaptive System, Institut d'Investigacions Biomediques August Pi i Sunyer, 08036, Barcelona, Spain. (7) Immunoreceptors of the Innate and Adaptive System, Institut d'Investigacions Biomediques August Pi i Sunyer, 08036, Barcelona, Spain. Servei d'Immunologia, Centre de Diagnostic Biomedic, Hospital Clinic de Barcelona, 08036, Barcelona, Spain. Departament de Biomedicina, Facultat de Medicina, Universitat de Barcelona, 08036, Barcelona, Spain.

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Programmed death-ligand 1 expression correlates with diminished CD8+ T cell infiltration and predicts poor prognosis in anal squamous cell carcinoma patients

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Objective: Increased expression of programmed death-ligand 1 (PD-L1) on tumor cells can be found in various malignancies; however, very limited information is known about its role in anal squamous cell carcinoma (ASCC). This study explored PD-L1 expression in ASCC patients and its association with patients' clinicopathological features, CD8+ T cell infiltration, and prognosis. Methods: Formalin-fixed paraffin-embedded tumor samples from 26 patients with ASCC were retrieved. The levels of PD-L1 expression on the membrane of both tumor cells and tumor-infiltrating mononuclear cells (TIMCs) were evaluated by immunohistochemistry. CD8+ T cell densities, both within tumors and at the tumor-stromal interface, were also analyzed. Baseline clinicopathological characteristics, human papilloma virus (HPV) status, and outcome data correlated with PD-L1-positive staining. Results: PD-L1 expression on tumor cells and TIMCs was observed in 46% and 50% of patients, respectively. Nineteen patients (73%) were HPV positive, with 7 showing PD-L1-positive staining on tumor cells and 9 showing PD-L1-positive staining on TIMCs. Increasing CD8+ density within tumors, but not immune stroma, was significantly associated with decreased PD-L1 expression by both tumor cells and TIMCs (P=0.0043 and P=0.0007). Patients with negative PD-L1 expression had significantly better progression-free survival (P=0.038 and P=0.0443) and a non-statistically significant trend toward longer overall survival (P=0.0882 and P=0.1222) compared with patients with positive PD-L1 expression. Conclusion: PD-L1 is widely expressed on the membrane of tumor cells and TIMCs in ASCCs. Its negative impact on prognosis may be due to the diminished CD8+ T cell infiltration within tumors.

Author Info: (1) Department of Colorectal Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center. (2)

Author Info: (1) Department of Colorectal Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center. (2) Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-sen University. (3) Department of Colorectal Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center. (4) Department of Colorectal Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center. (5) Department of Colorectal Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center. (6) Department of Colorectal Surgery, The Sixth Affiliated Hospital of Sun Yat-sen University. (7) Department of Pathology, Sun Yat-sen University Cancer Center, Guangzhou, People's Republic of China. (8) Department of Colorectal Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center. (9) Department of Colorectal Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center. (10) Department of Colorectal Surgery, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center.

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