Journal Articles

Immune monitoring

Techniques to monitor the immune response to cancer immunotherapy, to study immune cell interactions and to extend knowledge related to the induction and mechanisms of an immune response

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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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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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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Development of thyroid dysfunction is associated with clinical response to PD-1 blockade treatment in patients with advanced non-small cell lung cancer

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Purpose: Drugs that blockade interaction between programmed cell-death protein 1 (PD-1) and its ligand (PD-L1) are promising. Immune-related adverse events (irAEs) might be associated with favorable clinical outcomes, and thyroid dysfunction is one of the most common irAE. We evaluated the association of thyroid dysfunction during PD-1 blockade with the treatment efficacy in patients with non-small cell lung cancer (NSCLC). Experimental Design: A total 58 patients with stage IV NSCLC treated with PD-1 blockade were enrolled. Patients were categorized into thyroid dysfunction and euthyroid groups. Overall survival (OS) and progression-free survival (PFS) of the two groups were compared. Patients, tumor, and medication factors were adjusted using Cox proportional hazard modeling. Objective response rate (RR) and durable control rate were assessed according to the severity of thyroid dysfunction. Results: OS [median 118.0 (73.0-267.0) vs. 71.0 (28.0-160.0) days, log-rank P = 0.025] and PFS [118.0 (73.0-267.0) vs. 61.0 (28.0-130.0), log-rank P = 0.014] were longer in the thyroid dysfunction group. After adjustment, thyroid dysfunction was an independent predictive factor for favorable outcome [adjusted HR = 0.11 (95% CI) 0.01-0.92 for overall death; 0.38 (0.17-0.85) for disease progression]. The severity of thyroid dysfunction was associated with durable control rate (P for trend = 0.008). Conclusions: Thyroid dysfunction during PD-1 blockade is associated with treatment response and could provide supplementary information for immune monitoring in patients with advanced NSCLC.

Author Info: (1) Division of Endocrinology & Metabolism, Department of Medicine, Thyroid Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (2) Division of Endocrinology

Author Info: (1) Division of Endocrinology & Metabolism, Department of Medicine, Thyroid Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (2) Division of Endocrinology & Metabolism, Departments of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. (3) Division of Hematology and Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (4) Division of Endocrinology & Metabolism, Department of Medicine, Thyroid Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (5) Division of Endocrinology & Metabolism, Department of Medicine, Thyroid Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (6) Division of Endocrinology & Metabolism, Department of Medicine, Thyroid Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (7) Division of Endocrinology & Metabolism, Departments of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. (8) Division of Endocrinology & Metabolism, Departments of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. (9) Division of Endocrinology & Metabolism, Department of Medicine, Thyroid Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (10) Division of Endocrinology & Metabolism, Departments of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. (11) Division of Oncology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. (12) Division of Oncology, Department of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. (13) Division of Hematology and Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (14) Division of Hematology and Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (15) Division of Endocrinology & Metabolism, Department of Medicine, Thyroid Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea. (16) Division of Endocrinology & Metabolism, Departments of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. (17) Division of Endocrinology & Metabolism, Departments of Internal Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea. (18) Division of Endocrinology & Metabolism, Department of Medicine, Thyroid Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.

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Mutation load and an effector T-cell gene signature may distinguish immunologically distinct and clinically relevant lymphoma subsets

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Identifying follicular lymphoma (FL) patients with preexisting antitumor immunity will inform precision medicine strategies for novel cancer immunotherapies. Using clinical and genomic data from 249 FL patients, we determined the clinical impact of mutation load and an effector T-cell (Teff) gene signature as proxies for the likelihood of a functional immune response. The FL mutation load estimate varied between 0 and 33 mutations per Mb (median, 6.6), and 92% of FL patients with a high mutation load had high Teff gene expression (P = .001). The mutation load was associated with a benefit from rituximab maintenance: FL patients with low mutation loads experienced a profound benefit from rituximab maintenance (hazard ratio [HR], 0.29; 95% confidence interval [CI], 0.15-0.54; P < .001). The Teff gene signature was prognostic as a continuous predictor (P = .008), and was used to separate FL patients into 2 groups, an "inflamed" subset (Teff-high; n = 74) and an "uninflamed" subset (Teff-low; n = 75), with longer progression-free survival (PFS) in the inflamed FL subset (PFS HR, 0.39; 95% CI, 0.21-0.70; P = .002). Furthermore, the subset of inflamed FL tumors demonstrated high expression of other T-cell signatures and counterregulatory genes, which also correlate with PFS. Mutation load and Teff gene expression may help identify immunologically distinct lymphoma subsets relevant for modern immunotherapies.

Author Info: (1) Bioinformatics and. (2) Oncology Biomarker Development, Genentech, South San Francisco, CA. (3) Laboratory of Hematology, Lyon-Sud Hospital Center, Pierre-Benite, France. Cancer Research Center of

Author Info: (1) Bioinformatics and. (2) Oncology Biomarker Development, Genentech, South San Francisco, CA. (3) Laboratory of Hematology, Lyon-Sud Hospital Center, Pierre-Benite, France. Cancer Research Center of Lyon, INSERM U1052, Unite Mixte de Recherche Centre National de la Recherche Scientifique 5286, Lyon, France. (4) Foundation Medicine, Inc, Cambridge, MA. (5) Bioinformatics and. (6) Henri Becquerel Center, Rouen, France. (7) Gustave Roussy Institute, Villejuif, France; and. (8) Oncology Biomarker Development, Genentech, South San Francisco, CA. (9) Oncology Biomarker Development, Genentech, South San Francisco, CA. (10) Department of Bio-Pathology, Hematology, and Tumor Immunology, and. Paoli-Calmettes Institute, Aix-Marseille University, Marseille, France. (11) Laboratory of Hematology, Lyon-Sud Hospital Center, Pierre-Benite, France. Cancer Research Center of Lyon, INSERM U1052, Unite Mixte de Recherche Centre National de la Recherche Scientifique 5286, Lyon, France. (12) Laboratory of Hematology, Lyon-Sud Hospital Center, Pierre-Benite, France. Cancer Research Center of Lyon, INSERM U1052, Unite Mixte de Recherche Centre National de la Recherche Scientifique 5286, Lyon, France. (13) Oncology Biomarker Development, Genentech, South San Francisco, CA.

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High proportions of PD-1(+) and PD-L1(+) leukocytes in classical Hodgkin lymphoma microenvironment are associated with inferior outcome

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Immune checkpoint inhibition targeting the programmed death receptor (PD)-1 pathway is a novel treatment approach in relapsed and refractory classical Hodgkin lymphoma (cHL). Identifying patients with a high risk of treatment failure could support the use of PD-1 inhibitors as front-line treatment. Our aim was to investigate the prognostic impact of PD-1, programmed death-ligand 1 (PD-L1), and PD-L2 in the tumor microenvironment in diagnostic biopsies of patients with cHL. Patients from Denmark and Sweden, diagnosed between 1990 and 2007 and ages 15 to 86 years, were included. Tissue microarray samples were available from 387 patients. Immunohistochemistry was used to detect PD-1, PD-L1, and PD-L2, and the proportions of positive cells were calculated. Event-free survival (EFS; time to treatment failure) and overall survival (OS) were analyzed using Cox proportional hazards regression. High proportions of both PD-1(+) (hazard ratio [HR], 1.77; 95% confidence interval [CI], 1.10-2.86) and PD-L1(+) (HR = 1.89; 95% CI, 1.08-3.30) leukocytes in the microenvironment were associated with inferior EFS in a multivariate analysis (adjusted for white blood cell count >15 x 10(9)/L, hemoglobin <105 g/L, albumin <40 g/L, B symptoms, extranodal involvement, stage, bulky tumor, nodular sclerosis subtype, Epstein-Barr virus status, lymphocyte count <0.6 x 10(9)/L, sex, and country). A high proportion of PD-L1(+) leukocytes was also associated with inferior OS in a multivariate analysis (HR, 3.46; 95% CI, 1.15-10.37). This is the first study to show a correlation after multivariate analysis between inferior outcome in cHL and a high proportion of both PD-1(+) and PD-L1(+) leukocytes in the tumor microenvironment.

Author Info: (1) Experimental and Clinical Oncology, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. (2) Department of Hematology, Aarhus University Hospital, Aarhus, Denmark. (3)

Author Info: (1) Experimental and Clinical Oncology, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. (2) Department of Hematology, Aarhus University Hospital, Aarhus, Denmark. (3) Clinical Epidemiology Unit, Department of Medicine Solna, Karolinska Institute, Stockholm, Sweden. Hematology Center, Karolinska University Hospital, Stockholm, Sweden. (4) Experimental and Clinical Oncology, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. (5) Department of Hematology, Aarhus University Hospital, Aarhus, Denmark. (6) Department of Hematology, Aarhus University Hospital, Aarhus, Denmark. (7) Clinical and Experimental Pathology, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; and. (8) Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark. (9) Department of Hematology, Aarhus University Hospital, Aarhus, Denmark. (10) Experimental and Clinical Oncology, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. (11) Experimental and Clinical Oncology, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden. Clinical Epidemiology Unit, Department of Medicine Solna, Karolinska Institute, Stockholm, Sweden.

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Th17 immune microenvironment in Epstein-Barr virus-negative Hodgkin lymphoma: implications for immunotherapy

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Classical Hodgkin lymphoma (CHL) is a neoplasm characterized by robust inflammatory infiltrates and heightened expression of the immunosuppressive PD-1/PD-L1 pathway. Although anti-PD-1 therapy can be effective in >60% of patients with refractory CHL, improved treatment options are needed for CHLs which are resistant to anti-PD-1 or relapse after this form of immunotherapy. A deeper understanding of immunologic factors in the CHL microenvironment might support the design of more effective treatment combinations based on anti-PD-1. In addition, because the Epstein-Barr virus (EBV) residing in some CHL tumors is strongly immunogenic, we hypothesized that characteristics of the tumor immune microenvironment in EBV(+) CHL would be distinct from EBV(-) CHL, with specific implications for designing combination treatment regimens. Employing immunohistochemistry for immune cell subsets and checkpoint molecules, as well as gene expression profiling, we characterized 32 CHLs from the Johns Hopkins archives, including 12 EBV(+) and 20 EBV(-) tumors. Our results revealed a dichotomous cellular and cytokine immune milieu in EBV(+) vs EBV(-) CHL. EBV(+) tumors displayed a T helper 1 (Th1) profile typical of effective antitumor immunity, with increased infiltration of CD8(+) T cells and coordinate expression of the canonical Th1 transcription factor Tbet (TBX21), interferon-gamma (IFNG), and the IFN-gamma-inducible immunosuppressive enzyme indoleamine 2,3-dioxygenase. In contrast, EBV(-) tumors manifested a pathogenic Th17 profile and ongoing engagement of the interleukin-23 (IL-23)/IL-17 axis, with heightened phosphorylated signal transducer and activator of transcription 3 expression in infiltrating lymphocytes. These findings suggest that drugs blocking the IL-23/IL-17 axis, which are already in the clinic for treating certain autoimmune disorders, may enhance the therapeutic impact of anti-PD-1 therapy in EBV(-) CHL.

Author Info: (1) Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD. Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. (2) Bloomberg-Kimmel

Author Info: (1) Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD. Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. (2) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Oncology. (3) Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD. Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. (4) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Dermatology. (5) Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD. Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. (6) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Surgery, and. (7) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Surgery, and. (8) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Surgery, and. (9) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Dermatology. (10) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Dermatology. (11) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD. (12) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Oncology. (13) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Surgery, and. (14) Bloomberg-Kimmel Institute for Cancer Immunotherapy, Johns Hopkins University, Baltimore, MD; and. Department of Oncology.

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A library-based screening method identifies neoantigen-reactive T cells in peripheral blood prior to relapse of ovarian cancer

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Mutated cancer antigens, or neoantigens, represent compelling immunological targets and appear to underlie the success of several forms of immunotherapy. While there are anecdotal reports of neoantigen-specific T cells being present in the peripheral blood and/or tumors of cancer patients, effective adoptive cell therapy (ACT) against neoantigens will require reliable methods to isolate and expand rare, neoantigen-specific T cells from clinically available biospecimens, ideally prior to clinical relapse. Here, we addressed this need using "mini-lines", large libraries of parallel T cell cultures, each originating from only 2,000 T cells. Using small quantities of peripheral blood from multiple time points in an ovarian cancer patient, we screened over 3.3 x 10(6) CD8(+) T cells by ELISPOT for recognition of peptides corresponding to the full complement of somatic mutations (n = 37) from the patient's tumor. We identified ten T cell lines which collectively recognized peptides encoding five distinct mutations. Six of the ten T cell lines recognized a previously described neoantigen from this patient (HSDL1(L25V)), whereas the remaining four lines recognized peptides corresponding to four other mutations. Only the HSDL1(L25V)-specific T cell lines recognized autologous tumor. HSDL1(L25V)-specific T cells comprised at least three distinct clonotypes and could be identified and expanded from peripheral blood 3-9 months prior to the first tumor recurrence. These T cells became undetectable at later time points, underscoring the dynamic nature of the response. Thus, neoantigen-specific T cells can be expanded from small volumes of blood during tumor remission, making pre-emptive ACT a plausible clinical strategy.

Author Info: (1) Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada. Trev and Joyce Deeley Research Centre, British Columbia Cancer Agency, Victoria, British

Author Info: (1) Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada. Trev and Joyce Deeley Research Centre, British Columbia Cancer Agency, Victoria, British Columbia, Canada. Michael Smith's Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada. (2) Trev and Joyce Deeley Research Centre, British Columbia Cancer Agency, Victoria, British Columbia, Canada. (3) Trev and Joyce Deeley Research Centre, British Columbia Cancer Agency, Victoria, British Columbia, Canada. (4) Trev and Joyce Deeley Research Centre, British Columbia Cancer Agency, Victoria, British Columbia, Canada. Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada. (5) Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada. Michael Smith's Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada. Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada. (6) Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada. Trev and Joyce Deeley Research Centre, British Columbia Cancer Agency, Victoria, British Columbia, Canada. Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.

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Clinical response to PD-1 blockade correlates with a sub-fraction of peripheral central memory CD4+ T cells in patients with malignant melanoma

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Cancer immunotherapy that blocks immune checkpoint molecules, such as PD-1/PD-L1, unleashes dysfunctional antitumor T-cell responses and has durable clinical benefits in various types of cancers. Yet its clinical efficacy is limited to a small proportion of patients, highlighting the need for identifying biomarkers that can predict the clinical response by exploring antitumor responses crucial for tumor regression. Here we explored T-cell responses associated with clinical benefits using peripheral blood mononuclear cells (PBMCs) from patients with malignant melanoma treated with anti-PD-1 monoclonal antibody (mAb). Pre- and post-treatment samples were collected from two different cohorts (discovery set and validation set) and subjected to mass cytometry assays that measured the expression levels of 35 proteins. Screening by high dimensional clustering in the discovery set identified increases in three micro-clusters of CD4+ T cells, a subset of central memory CD4+ T cells harboring CD27+FAS-CD45RA-CCR7+ phenotype, after treatment in long-term survivors, but not in non-responders. The same increase was also observed in clinical responders in the validation set. We propose that increases in this subset of central memory CD4+ T cells in peripheral blood can be potentially used as a predictor of clinical response to PD-1 blockade therapy in patients with malignant melanoma.

Author Info: (1) Division of Cancer Immunology, Research Institute, National Cancer Center, Tokyo/Chiba, Japan. Division of Cancer Immunology, Exploratory Oncology Research & Clinical Trial Center (EPOC), National

Author Info: (1) Division of Cancer Immunology, Research Institute, National Cancer Center, Tokyo/Chiba, Japan. Division of Cancer Immunology, Exploratory Oncology Research & Clinical Trial Center (EPOC), National Cancer Center, Tokyo/Chiba, Japan. Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. (2) Department of Dermatology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. (3) Division of Cancer Immunology, Research Institute, National Cancer Center, Tokyo/Chiba, Japan. Division of Cancer Immunology, Exploratory Oncology Research & Clinical Trial Center (EPOC), National Cancer Center, Tokyo/Chiba, Japan. (4) Department of Dermatology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. (5) Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. (6) Division of Cancer Immunology, Research Institute, National Cancer Center, Tokyo/Chiba, Japan. Division of Cancer Immunology, Exploratory Oncology Research & Clinical Trial Center (EPOC), National Cancer Center, Tokyo/Chiba, Japan. Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Japan.

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