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PubMed | Karolinska Institutet, Janssen Research & Development LLC, Nodality, Omni Array Biotechnology and 8 more.
Type: | Journal: Journal for immunotherapy of cancer | Year: 2016

There is growing recognition that immunotherapy is likely to significantly improve health outcomes for cancer patients in the coming years. Currently, while a subset of patients experience substantial clinical benefit in response to different immunotherapeutic approaches, the majority of patients do not but are still exposed to the significant drug toxicities. Therefore, a growing need for the development and clinical use of predictive biomarkers exists in the field of cancer immunotherapy. Predictive cancer biomarkers can be used to identify the patients who are or who are not likely to derive benefit from specific therapeutic approaches. In order to be applicable in a clinical setting, predictive biomarkers must be carefully shepherded through a step-wise, highly regulated developmental process. Volume I of this two-volume document focused on the pre-analytical and analytical phases of the biomarker development process, by providing background, examples and good practice recommendations. In the current Volume II, the focus is on the clinical validation, validation of clinical utility and regulatory considerations for biomarker development. Together, this two volume series is meant to provide guidance on the entire biomarker development process, with a particular focus on the unique aspects of developing immune-based biomarkers. Specifically, knowledge about the challenges to clinical validation of predictive biomarkers, which has been gained from numerous successes and failures in other contexts, will be reviewed together with statistical methodological issues related to bias and overfitting. The different trial designs used for the clinical validation of biomarkers will also be discussed, as the selection of clinical metrics and endpoints becomes critical to establish the clinical utility of the biomarker during the clinical validation phase of the biomarker development. Finally, the regulatory aspects of submission of biomarker assays to the U.S. Food and Drug Administration as well as regulatory considerations in the European Union will be covered.


PubMed | Karolinska Institutet, Janssen Research & Development LLC, Nodality, Omni Array Biotechnology and 8 more.
Type: | Journal: Journal for immunotherapy of cancer | Year: 2016

Immunotherapies have emerged as one of the most promising approaches to treat patients with cancer. Recently, there have been many clinical successes using checkpoint receptor blockade, including T cell inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death-1 (PD-1). Despite demonstrated successes in a variety of malignancies, responses only typically occur in a minority of patients in any given histology. Additionally, treatment is associated with inflammatory toxicity and high cost. Therefore, determining which patients would derive clinical benefit from immunotherapy is a compelling clinical question. Although numerous candidate biomarkers have been described, there are currentlythree FDA-approved assays based on PD-1 ligand expression (PD-L1) that have been clinically validated toidentify patients whoare more likely to benefit from asingle-agent anti-PD-1/PD-L1 therapy. Because of the complexity of the immune response and tumor biology, it is unlikely that a single biomarker will be sufficient to predict clinical outcomes in response to immune-targeted therapy. Rather, the integration of multiple tumor and immune response parameters, such as protein expression, genomics, and transcriptomics, may be necessary for accurateprediction of clinicalbenefit. Before a candidate biomarker and/or new technology can be used in a clinical setting, several steps are necessary to demonstrate its clinical validity. Although regulatory guidelines provide general roadmaps for the validation process, their applicability to biomarkers in the cancer immunotherapy field is somewhat limited. Thus, Working Group 1 (WG1) of the Society for Immunotherapy of Cancer (SITC) Immune Biomarkers Task Force convened to address this need. In this two volume series, we discuss pre-analytical and analytical (Volume I) as well as clinical and regulatory (Volume II) aspects of the validation process as applied to predictive biomarkers for cancer immunotherapy. To illustrate the requirements for validation, wediscuss examples of biomarker assays that have shown preliminary evidence of an association with clinical benefit from immunotherapeutic interventions. The scope includes only those assays and technologies that have established a certain level of validation for clinical use (fit-for-purpose). Recommendations to meet challenges and strategies to guide the choice of analytical and clinical validation design for specific assays are also provided.


Van Der Burg S.H.,Leiden University | Kalos M.,University of Pennsylvania | Gouttefangeas C.,University of Tübingen | Janetzki S.,ZellNet Consulting Inc. | And 6 more authors.
Science Translational Medicine | Year: 2011

Assays that measure a patient's immune response play an increasingly important role in the development of immunotherapies. The inherent complexity of these assays and independent protocol development between laboratories result in high data variability and poor reproducibility. Quality control through harmonization - based on integration of laboratory-specific protocols with standard operating procedures and assay performance benchmarks - is one way to overcome these limitations. Harmonization guidelines can be widely implemented to address assay performance variables. This process enables objective interpretation and comparison of data across clinical trial sites and also facilitates the identification of relevant immune biomarkers, guiding the development of new therapies.


Janetzki S.,ZellNet Consulting Inc. | Price L.,LBPrice Statistical Consulting Ltd. | Schroeder H.,BioNTech Diagnostics GmbH | Britten C.M.,Association for Cancer Immunotherapy CIMT | And 3 more authors.
Nature Protocols | Year: 2015

The presented protocol for Elispot plate evaluation summarizes how to implement the recommendations developed following the establishment of a large-scale international Elispot plate-reading panel and subsequent multistep consensus-finding process. The panel involved >100 scientists from various immunological backgrounds. The protocol includes the description and justification of steps for setting reading parameters to obtain accurate, reliable and precise automated analysis results of Elispot plates. Further, necessary adjustments for out-of-specification situations are described and examples are provided. The plate analysis, including parameter adjustments, auditing of results and necessary annotations, should be achievable within a time range of 10-30 min per plate. Adoption of these guidelines should enable a further reduction in assay variability and an increase in the reliability and comparability of results obtained by Elispot. These guidelines conclude the ongoing harmonization efforts for the enzymatic Elispot assay. © 2015 Nature America, Inc. All rights reserved.


PubMed | Leiden University, LBPrice Statistical Consulting Ltd., Collegeville, ZellNet Consulting Inc. and 2 more.
Type: Journal Article | Journal: Nature protocols | Year: 2015

The presented protocol for Elispot plate evaluation summarizes how to implement the recommendations developed following the establishment of a large-scale international Elispot plate-reading panel and subsequent multistep consensus-finding process. The panel involved >100 scientists from various immunological backgrounds. The protocol includes the description and justification of steps for setting reading parameters to obtain accurate, reliable and precise automated analysis results of Elispot plates. Further, necessary adjustments for out-of-specification situations are described and examples are provided. The plate analysis, including parameter adjustments, auditing of results and necessary annotations, should be achievable within a time range of 10-30 min per plate. Adoption of these guidelines should enable a further reduction in assay variability and an increase in the reliability and comparability of results obtained by Elispot. These guidelines conclude the ongoing harmonization efforts for the enzymatic Elispot assay.


Moodie Z.,Fred Hutchinson Cancer Research Center | Price L.,New York University | Janetzki S.,ZellNet Consulting Inc. | Britten C.M.,Johannes Gutenberg University Mainz
Methods in Molecular Biology | Year: 2012

ELISPOT assay readout is often dichomized as positive or negative responses according to prespecified criteria. However, these criteria can vary widely across institutions. The adoption of a common response criterion is a key step toward cross-laboratory comparability. This chapter describes the two main approaches to response determination, identifying the strengths and limitations of each. Nonparametric statistical tests and consideration of data quality are recommended and instructions provided for their ready implementation by nonstatisticians and statisticians alike. © 2012 Springer Science+Business Media, LLC.


Janetzki S.,ZellNet Consulting Inc. | Britten C.M.,Johannes Gutenberg University Mainz
Methods in Molecular Biology | Year: 2012

During more than 25 years of application in immunological sciences, ELISPOT has been established as a routine, robust, versatile, and reliable assay. From basic research to clinical immune monitoring, ELISPOT is being used to address the quantification and (to a lesser extent) functional characterization of immune cells secreting different molecules in the context of health and disease, immune intervention, and therapy in humans and other species [Kalyuzhny (Ed.) (2005) Handbook of Elispot: methods and protocols, Vol. 302, Humana Press Inc., Totowa, NJ]. Over the last decade, ELISPOT assays have been increasingly implemented as an immune-monitoring tool in clinical trials [Schmittel et al. J Immunother 23:289-295, 2000; Whiteside Immunol Invest 29:149-162, 2000; Nagata et al. Ann N Y Acad Sci 1037:10-15, 2004; Cox et al. (2005) Cellular immune assays for evaluation of vaccine efficacy in developing countries., In Manual of Clinical Immunology Laboratory (Rose, N. R., Hamilton, R. G., and Detrick, B., Eds.), p 301, ASM Press, Washington, DC; Cox et al. Methods 38:274-282, 2006]. While the principles of the original protocol have changed little since its first introduction [Czerkinsky J Immunol Methods 110:29-36, 1988], individual laboratories have adapted assay procedures based on experimental needs, availability of reagents and equipment, obtained recommendations, and gained experience, leading to a wide disparity of applied ELISPOT protocols with inevitable consequences. This chapter addresses the resulting challenges for ELISPOT use in clinical trial settings, and discusses the influence of harmonization strategies as a tool for overcoming these challenges. Furthermore, harmonization is discussed in the context of assay standardization and validation strategies. © 2012 Springer Science+Business Media, LLC.


Filbert H.,Johannes Gutenberg University Mainz | Attig S.,Johannes Gutenberg University Mainz | Bidmon N.,Johannes Gutenberg University Mainz | Renard B.Y.,Johannes Gutenberg University Mainz | And 8 more authors.
Cancer Immunology, Immunotherapy | Year: 2013

Robust and sensitive ELISPOT protocols are commonly applied concomitant with the development of new immunotherapeutics. Despite the knowledge that individual serum batches differ in their composition and may change properties over time, serum is still commonly used in immunologic assays. Commercially available serum batches are expensive, limited in quantity and need to be pretested for suitability in immunologic assays, which is a laborious process. The aim of this study was to test whether serum-free freezing media can lead to high cell viability and favorable performance across multiple ELISPOT assay protocols. Thirty-one laboratories from ten countries participated in a proficiency panel organized by the Cancer Immunotherapy Immunoguiding Program to test the influence of different freezing media on cell quality and immunologic function. Each center received peripheral blood mononuclear cells which were frozen in three different media. The participants were asked to quantify antigen-specific CD8+ T-cell responses against model antigens using their locally established IFN-gamma ELISPOT protocols. Self-made and commercially available serum-free freezing media led to higher cell viability and similar cell recovery after thawing and resting compared to freezing media supplemented with human serum. Furthermore, the test performance as determined by (1) background spot production, (2) replicate variation, (3) frequency of detected antigen-specific spots and (4) response detection rate was similar for serum and serum-free conditions. We conclude that defined and accessible serum-free freezing media should be recommended for freezing cells stored for subsequent ELISPOT analysis. © 2012 The Author(s).


Moodie Z.,Fred Hutchinson Cancer Research Center | Price L.,New York University | Gouttefangeas C.,University of Tübingen | Mander A.,University of Southampton | And 7 more authors.
Cancer Immunology, Immunotherapy | Year: 2010

No consensus has been reached on how to determine if an immune response has been detected based on raw data from an ELISPOT assay. The goal of this paper is to enable investigators to understand and readily implement currently available methods for response determination. We describe empirical and statistical approaches, identifying the strengths and limitations of each approach to allow readers to rationally select and apply a scientifically sound method appropriate to their specific laboratory setting. Five representative approaches were applied to data sets from the CIMT Immunoguiding Program and the response detection and false positive rates were compared. Simulation studies were also performed to compare empirical and statistical approaches. Based on these, we recommend the use of a non-parametric statistical test. Further, we recommend that six medium control wells or four wells each for both medium control and experimental conditions be performed to increase the sensitivity in detecting a response, that replicates with large variation in spot counts be filtered out, and that positive responses arising from experimental spot counts below the estimated limit of detection be interpreted with caution. Moreover, a web-based user interface was developed to allow easy access to the recommended statistical methods. This interface allows the user to upload data from an ELISPOT assay and obtain an output file of the binary responses. © 2010 The Author(s).


PubMed | ZellNet Consulting Inc.
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2015

The ELISpot, a heterogeneous immunoassay, is widely used for detection of low abundant analytes. It is a reliable and robust assay to monitor responses of the immune system at the single-cell level by capturing secreted molecules of interest with specific, membrane-bound antibodies. Those molecules are then made visible by a cascade of ELISA-related development steps. The final results are distinct spots on the membrane as an imprint of the cell secreting the captured molecules, not only allowing their quantification but also providing insight on the kinetics and strength of secretion. This chapter describes the optimized protocol steps of the ELISpot technique, important improvements and tools available for the community, and the current expansion of the technique into polyfunctional cell analysis.

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