Abstract 6296: Multiplexed protein and RNA quantification on a single instrument harmonizes multi-omic analyses of biomarkers for immunotherapies and targeted therapies in non-small cell lung cancer
Authors: Khan Yasef , Ramirez Francisco , Gokul Shobha , Manzano Lawrence , Leong Louis , Latham Gary J. , Heger Chris 
Published: September 2020

Introduction: Integration of multiple classes of biomarkers, including proteins, DNA and RNA, offers insights into the complexities of spatial, temporal, and functional biology beyond those possible with any one class. The distinct properties and detection modalities of protein and nucleic acid, however, require different technologies and platforms, thereby limiting their utility in a single-assay format. We developed a multiplexed multi-omic workflow using an established protein analysis system (Simple Western, ProteinSimple) to assess both proteins and nucleic acids. We demonstrate that this system and workflow can quantify at least six well-established proteins and RNA biomarkers for immunotherapies and targeted therapies for non-small cell lung cancer (NSCLC).

Methods: The multi-omic workflow required only three steps. First, protein and RNA were extracted from cell lines or clinical FFPE tumor biopsies. Second, hapten-labeled amplicons were prepared using single-tube multiplex RT-PCR. Finally, both amplicons and proteins were separated on a Simple Western instrument, detected by immunoassay, and analyzed. RNA quantification via RT-PCR was normalized to either total RNA or a stable reference gene, and compared with multiple orthogonal assays, including nucleic acid fragment capillary electrophoresis (CE) system and RT-qPCR.

Results: PCR or RT-PCR amplicons from EML4-ALK RNA fusions, PD-L1 and PD-L2 transcripts, and a reference mRNA were designed with varying amplicon lengths for CE separation optimization. RNA-based products were quantified with a linear dynamic range of ~2 logs using Simple Western. The relative expression of PD-L1 RNA in four cancer cells was consistent using this approach compared to two other CE systems and qPCR. Further, the results matched published data (rank order A549 &lt; PC9 &lt; H460 &lt; H441). PD-L1 protein levels assessed on the same system were also consistent with literature data. EML4-ALK expression agreed with known results for both ALK-positive and negative cell lines. All results were then recapitulated in a single-workflow, 6-plex RNA and protein assay. Finally, multiplexed protein and RNA expression of NSCLC FFPE tumor biopsies was measured simultaneously and compared.

Conclusions: We demonstrate that a technology (Simple Western) originally developed for protein analysis is capable of analyzing nucleic acids. We also show the potential utility of this system to quantify protein and RNA biomarkers relevant to immune checkpoint blockade and targeted therapies, as shown for NSCLC. This novel approach can combine protein, DNA and RNA quantification in one harmonized, multiplexed workflow with reduced sample requirements, and decreased inter-assay variability, with a more streamlined and less error-prone workflow.

Citation Format: Yasef Khan, Francisco Ramirez, Shobha Gokul, Lawrence Manzano, Louis Leong, Gary J. Latham, Chris Heger. Multiplexed protein and RNA quantification on a single instrument harmonizes multi-omic analyses of biomarkers for immunotherapies and targeted therapies in non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6296.