Abstract 3213: A rapid, sensitive and quantitative method to measure protein biomarkers MUC16 and MSLN from archived clinical samples using TaqMan proximity ligation assays

Autor: Aimee Fourie, An Do, Yulei Wang, Kathy Kozak, Suzie J. Scales, Lisa Ryner, Ron Firestein, Siao Ping Tsai, Yinghui Guan
Rok vydání: 2013
Předmět:
Zdroj: Cancer Research. 73:3213-3213
ISSN: 1538-7445
0008-5472
DOI: 10.1158/1538-7445.am2013-3213
Popis: MUC16 and MSLN are tumor-specific antigens highly prevalent in ovarian and pancreatic cancers and have been used as targets in developing antibody-drug conjugates (ADCs). Development of a sensitive method to quantitatively measure these protein biomarkers in archival clinical samples may enable potential companion diagnostics to these novel therapeutic agents. Here we report the development of a method to quantitatively measure these biomarkers from formalin fixed paraffin embedded (FFPE) tumor samples using TaqMan proximity ligation assays (PLA). Monoclonal antibodies that recognize the extracellular domains of MUC16 and MSLN were conjugated to amplicon forming oligonucleotides to generate proximity ligation probes for TaqMan quantification. Using purified MUC16 and MSLN recombinant protein the assay sensitivities were determined to be lower than single-digit ng/mL concentrations for both MUC16 and MSLN and the dynamic ranges were >3 logs and ∼2 logs, respectively. In addition, the assays were demonstrated to be highly selective by testing lysates from expressing and non-expressing cell lines as determined by qRT-PCR. For clinical application of these assays, we further developed and optimized a simple two-step method for extraction and antigen retrieval of protein from archival FFPE tissues. FFPE extracts were prepared from a set of 10um sections of ovarian tumor tissue blocks and analyzed to determine relative MUC16 and MSLN protein expression levels. We benchmarked these results from the FFPE protocol with PLA data generated from matched fresh frozen tumor tissue as well as expression level determined by immunohistochemistry (IHC) and gene expression level determined by qRT-PCR assays from the same FFPE tissue blocks. All three comparisons with FFPE PLA were remarkably well correlated. In conclusion, we have established the technical feasibility and clinical utility of TaqMan PLA-based protein assays for sensitive and quantitative measurement of protein biomarkers in archival clinical samples. The methodology developed in this study represents a promising approach in companion diagnostic development for quantitative protein biomarker assessment. Citation Format: Lisa Ryner, Aimee Fourie, Siao Ping Tsai, Suzie Scales, Kathy Kozak, Ron Firestein, Yinghui Guan, An Do, Yulei Wang. A rapid, sensitive and quantitative method to measure protein biomarkers MUC16 and MSLN from archived clinical samples using TaqMan proximity ligation assays. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3213. doi:10.1158/1538-7445.AM2013-3213
Databáze: OpenAIRE
Externí odkaz: