Abstract 5279: Establishing robust pharmacodynamic (PD) immunofluorescence assays of clinical biopsies at the National Cancer Institute: Optimized quality control procedures for the evaluation of DNA damage response and epithelial-mesenchymal transition (EMT) biomarkers

Autor: Katherine V. Ferry-Galow, Tony Navas, Scott M. Lawrence, Robert J. Kinders, Donna Butcher, Brad A. Gouker, Jiuping Ji, Joseph E. Tomaszewski, Hala R. Makhlouf, Ralph E. Parchment, William H. Yutzy, James H. Doroshow, Shivaani Kummar
Rok vydání: 2015
Předmět:
Zdroj: Cancer Research. 75:5279-5279
ISSN: 1538-7445
0008-5472
DOI: 10.1158/1538-7445.am2015-5279
Popis: Robust PD assay results are valuable for informing decisions about continued preclinical and clinical development of new agents and for identifying effective combinations of targeted agents. The National Cancer Institute's Division of Cancer Treatment and Diagnosis (DCTD) develops and validates PD assays to obtain accurate information about drug effect on intended molecular targets in first-in-human clinical trials. Our group utilizes quantitative immunofluorescence assays (qIFA) of PD biomarkers in FFPE slides prepared from pre- and post-dose tumor biopsies collected from patients on early phase clinical trials. Paired biopsies are fixed and paraffin embedded, in parallel with tissue controls, such that all tissues are sectioned onto the same slide. Stringent methods to maintain the biopsy orientation are utilized during fixation, blocking and microtomy. A large number of sections are generated to increase the likelihood of finding optimal tumor regions of interest for biomarker analyses. Flanking H&E slides are utilized to assess the quality of the tumor biopsies. Whole slide scans of the H&E slides are shared with a clinical pathologist who determines whether the sections are sufficient or insufficient for the intended analysis. The pathology review allows the assay operator to select the optimal range of slides to stain and quantitatively analyze for the biomarker(s) of interest. Pathology-guided regions allow the operator to focus on tumor regions of interest and avoid normal tissue and/or confounding regions compromised by handling and processing artifacts. For nuclear biomarkers such as γH2AX and related DNA damage response biomarkers the pathologist annotates areas of sufficient tumor content and viability. For biomarker changes in tissue architecture such as that seen in Epithelial Mesenchymal Transition biomarkers, including E-Cadherin, Vimentin and β-catenin, the pathology annotation denotes areas of normal tissue and necrosis to eliminate from the qIFA analysis. All other areas of sufficient tumor cellularity from the entire slide are evaluated for the EMT biomarkers. Use of the pathology-annotated whole slide image as a tool for guidance of the operator performing the quantitative evaluation of the PD biomarker helps to ensure a non-subjective analysis. Finally, steps are taken to ensure proper storage of the paraffin slides, including paraffin dipping after microtomy, which is critical for preservation of labile epitopes such as phosphorylated proteins. We will present details of these optimized methods as well as key lessons learned during the preclinical and clinical implementation of qIFA measurements of PD biomarkers for various molecular targeted agents. Funded by NCI Contract No HHSN261200800001E. Citation Format: Katherine V. Ferry-Galow, Scott M. Lawrence, Tony Navas, Hala R. Makhlouf, Donna O. Butcher, Brad A. Gouker, William H. Yutzy, Jiuping Ji, Robert Kinders, Ralph E. Parchment, Shivaani Kummar, Joseph E. Tomaszewski, James H. Doroshow. Establishing robust pharmacodynamic (PD) immunofluorescence assays of clinical biopsies at the National Cancer Institute: Optimized quality control procedures for the evaluation of DNA damage response and epithelial-mesenchymal transition (EMT) biomarkers [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5279. doi:10.1158/1538-7445.AM2015-5279
Databáze: OpenAIRE
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