Abstract 918: Clinical validation of a multiplexed ChemoPlex SRM assay in FFPE human tumor tissue
Authors: An Eunkyung , Liao Wei-Li , Thyparambil Sheeno , Blackler Adele , Uzzell Jamar , Bengali Kathleen , Darfler Marlene , Burrows Jon , Hembrough Todd 
Published: May 2015

Abstract
Background: Current cancer treatment regimens rely on the use of chemotherapy agents that inhibit DNA replication and repair machinery. Several proteins are involved in this mechanism, such as TOPO1, TOPO2A, RRM1, FR-alpha and hENT1. The expression levels and activities of these proteins can greatly affect the success of chemotherapy; however current treatment indications are not based on tumor expression levels of these proteins. We have developed a quantitative, multiplexed ChemoPlex SRM method to evaluate these markers in a host of solid tumors from a limited amount of FFPE biopsy tissue using our Liquid Tissue®-SRM (LT-SRM) platform. Use of this method will enable a physician to understand individual tumor molecular machinery and ultimately could lead to individualized treatment decisions leading to better patient care.
Methods: We used trypsin digestion mapping of recombinant proteins to identify optimal quantitative peptides for the ChemoPlex SRM assay. Standard curves were generated to determine the LOD, LOQ, accuracy, precision and linearity of the assay. The assay was pre-clinically validated on 14 cell lines with known expression levels of these Chemo-targets, and the assay was then run on microdissected archived FFPE human tissue samples from lung, gastro-esophageal cancer (GEC), breast, liver, colorectal, and ovarian tumors.
Results: The peptides chosen for the 5 Chemo-Plex targets had LOD values of 150, 50, 300, 200, and 100 amol (CV&lt;20%) for FR alpha, hENT1, TOPO1, TOPO2A, and RRM1, respectively. Fourteen cell lines were assayed for the Chemo-target expressions by LT-SRM, and regression analysis between protein and mRNA analysis for each target demonstrated varying correlations (R2=0.91(FR alpha); 0.78 (hENT1); 0.16 (TOPO1); 0.56 (TOPO2A); 0.59 (RRM1)) suggesting that RT-PCR measurements of mRNA levels would not be representative of cellular protein levels and therefore not useful for biomarker analysis for physicians. Our initial clinical analysis shows that FR alpha was detectable only in certain lung tumors (especially adenocarcinoma) and ovarian tumors; breast cancer tumors were found to have a wide range of hENT1 expression (LOD -1,284 amol/ug) while hENT1 expression in other tissues ranged from 90 to 377 amol/ug. TOPO1 had fairly ubiquitous expression (359 -1,300 amol/ug) except for one GEC and one breast cancer tissue. TOPO2A was identified in all tissue types (227-1,057 amol/ug). All samples except 1 GEC tissue express RRM1 (160 - 958 amol/ug)
Discussion: We describe the development and initial clinical validation of a quantitative proteomic ChemoPlex SRM assay which accurately measures the expression of five chemotherapy targets in FFPE tumor tissue. When multiplexed along with other druggable biomarkers, the ChemoPlex SRM assay will allow more accurate identification of patients that are likely to benefit from the combination of chemotherapy and targeted therapies.
Citation Format: Eunkyung An, Wei-Li Liao, Sheeno Thyparambil, Adele Blackler, Jamar Uzzell, Kathleen Bengali, Marlene Darfler, Jon Burrows, Todd Hembrough. Clinical validation of a multiplexed ChemoPlex SRM assay in FFPE human tumor tissue. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 918. doi:10.1158/1538-7445.AM2014-918