Translating pharmacodynamic biomarkers from bench to bedside: analytical validation and fit-for-purpose studies to qualify multiplex immunofluorescent assays for use on clinical core biopsy specimens

Marrero, Allison M.; Lawrence, Scott M.; Wilsker, Deborah; Voth, Andrea Regier; Kinders, Robert J.

doi:10.1053/j.seminoncol.2016.06.003

Cited by 11 publications

(9 citation statements)

References 32 publications

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“…Multiplexing also reduces the possibility of missing a pharmacodynamic response due to specimen timing, dose schedule, or genetic alterations in the tumor (compared with a single biomarker readout). For instance, the lack of modulation at a given time point in a single analyte assay could be wrongly interpreted as either no drug effect or a genetic defect that prevents modulation of the target, a risk that can be circumvented by multiplexing (40). The importance of this approach is illustrated by the treatment of A673 xenografts with gemcitabine ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers

Wilsker

Barrett

Dull

et al. 2019

Clinical Cancer Research

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Purpose: We sought to examine the pharmacodynamic activation of the DNA damage response (DDR) pathway in tumors following anticancer treatment for confirmation of target engagement. Experimental Design: We evaluated the time course and spatial activation of 3 protein biomarkers of DNA damage recognition and repair (gH2AX, pS343-Nbs1, and Rad51) simultaneously in a quantitative multiplex immunofluorescence assay (IFA) to assess DDR pathway activation in tumor tissues following exposure to DNA-damaging agents. Results: Because of inherent biological variability, baseline DDR biomarker levels were evaluated in a colorectal cancer microarray to establish clinically relevant thresholds for pharmacodynamic activation. Xenograft-bearing mice and clinical colorectal tumor biopsies obtained from subjects exposed to DNA-damaging therapeutic regimens demonstrated marked intratumor heterogeneity in the timing and extent of DDR biomarker activation due, in part, to the cell-cycle dependency of DNA damage biomarker expression. Conclusions: We have demonstrated the clinical utility of this DDR multiplex IFA in preclinical models and clinical specimens following exposure to multiple classes of cytotoxic agents, DNA repair protein inhibitors, and molecularly targeted agents, in both homologous recombinationproficient and-deficient contexts. Levels exceeding 4% nuclear area positive (NAP) gH2AX, 4% NAP pS343-Nbs1, and 5% cells with !5 Rad51 nuclear foci indicate a DDR activation response to treatment in human colorectal cancer tissue. Determination of effect-level cutoffs allows for robust interpretation of biomarkers with significant interpatient and intratumor heterogeneity; simultaneous assessment of biomarkers induced at different phases of the DDR guards against the risk of false negatives due to an ill-timed biopsy.

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Section: Discussionmentioning

confidence: 99%

Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers

Wilsker

Barrett

Dull

et al. 2019

Clinical Cancer Research

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“…Feasibility studies are often necessary prior to incorporating biomarkers into clinical trials, or in settings where the tissue analyzed is irreplaceable. Standard operating procedures and quality assurance protocols must be developed that consider accuracy, precision, sensitivity and specificity, and turnaround time of assessments must be within acceptable timeframes (52–54). …”

Section: The Search For Valuable Immuno-oncology Biomarkers Requires mentioning

confidence: 99%

“…The information provided by these techniques may reveal insights into the cell-cell interactions that are important for response to immune modulating agents and provide novel biomarkers for study in clinical trials. Particular attention to reagent validation and assay calibration is necessary to carry out these techniques, and the impact of tissue quantity and quality (i.e., liquid or small, fine needle aspirate biopsies versus core biopsies) cannot be underestimated (61,62). …”

Section: The Search For Valuable Immuno-oncology Biomarkers Requires mentioning

confidence: 99%

The Challenge for Development of Valuable Immuno-oncology Biomarkers

Mehnert

Monjazeb

Beerthuijzen

et al. 2017

Clinical Cancer Research

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The development of immunotherapy is an important breakthrough for the treatment of cancer, with anti-tumor efficacy observed in a wide variety of tumors. To optimize immunotherapy use, approaches must be developed to identify which patients are likely to achieve benefit. To minimize therapeutic toxicities and costs, understanding the ideal choice and sequencing of the numerous immuno-oncology agents available for individual patients is thus critical, but fraught with challenges. The immune tumor microenvironment (TME) is a unique aspect of the response to immuno-oncology agents and measurement of single biomarkers does not adequately capture these complex interactions. Therefore, multiple potential biomarkers are likely needed. Current candidates in this area include PD-L1 expression, CD8+ tumor infiltrating lymphocytes, tumor mutation load and neoantigen burden, immune related gene signatures and multiplex immunohistochemical assays that examine the pharmacodynamic and spatial interactions of the TME. The most fruitful investigations are likely to use several techniques to predict response and interrogate mechanisms of resistance. Immuno-oncology biomarker research must employ validated assays to ask focused research questions utilizing clinically annotated tissue collections and biomarker focused clinical trial designs to investigate specific endpoints. Real time input from patients and their advocates into biomarker discovery is necessary to ensure that the investigations pursued will improve both clinical outcomes and quality of life. We herein provide a framework of recommendations to guide the search for immuno-oncology biomarkers of value.

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“…particularly when incorporating open-source or proprietary imaging analysis tools, (Alegro et al, 2017;Camp et al, 2002;Marrero et al, 2016;McCabe et al, 2005). Although our protocol accommodates up to 11 markers, this number could be possibly increased in labs with access to fluorescent microscopes equipped with more than four channels.…”

Section: Discussionmentioning

confidence: 99%

Multiplex immunofluorescence methods in neurodegenerative diseases

Ehrenberg

Morales

Tejedor

et al. 2019

Preprint

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BACKGROUND: Neurodegenerative diseases feature stereotypical deposits of protein aggregates that selectively accumulate in vulnerable cells. The ability to simultaneously localize multiple targets in situ would facilitate discovery and validation of molecular pathways underlying neurodegenerative diseases.Immunostaining methods offer in situ detection of targets. Most proposed protocols for multiplexing immunostaining require sophisticated equipment not available for standard labs. Effective stripping of antibodies, allowing successive rounds of staining while maintaining tissue and antigen integrity, is the main roadblock for enabling multiplex immunostaining in standard labs. Antibody stripping techniques have not been fully validated for neurodegenerative disease research. Moreover, despite the increased complexity of multiplex immunostaining protocols, quality control steps have not been regularly implemented.NEW METHOD: Aiming to create protocols for multiplex localization of neurodegenerative-related processes, without the need for specialized equipment, we evaluated several antibody stripping techniques. We also recommend quality control steps to validate stripping efficacy and ameliorate concerns of cross-reactivity and false positives. RESULTS:The proposed protocol using β -mercaptoethanol enables reliable antibody stripping across multiple rounds of staining and minimizes the odds of cross-reactivity while preserving tissue and antigen integrity.COMPARISON WITH EXISTING METHODS: Our proposed method accounts for the intricacies of suboptimal human post-mortem tissue and the need to strip markers bound to highly aggregated proteins.Additionally, it incorporates quality control steps to validate antibody stripping.CONCLUSIONS: Multiplex immunofluorescence methods for studying neurodegenerative diseases in human postmortem tissue are feasible even in standard laboratories. Nevertheless, evaluation of stripping parameters in optimization and validation phases of experiments is prudent.

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Translating pharmacodynamic biomarkers from bench to bedside: analytical validation and fit-for-purpose studies to qualify multiplex immunofluorescent assays for use on clinical core biopsy specimens

Cited by 11 publications

References 32 publications

Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers

Evaluation of Pharmacodynamic Responses to Cancer Therapeutic Agents Using DNA Damage Markers

The Challenge for Development of Valuable Immuno-oncology Biomarkers

Multiplex immunofluorescence methods in neurodegenerative diseases

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