Abstract 1469: Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTM and NanoStringTM from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors
Authors: Au Qingyan , Padmanabhan RaghavKrishna , Tran Nam , Chandramohan Lakshmi , Hoe Nicholas 
Published: July 2017

Abstract
Cancer is characterized as a loss of normal cellular regulation, due to accumulation of mutations and disruption of complex biological pathways. MicroRNAs (miRNAs) regulation of co-stimulatory and immune checkpoint pathways have been implicated as one of the potential mechanisms for cancer evasion in immuno-oncology. It is estimated that 30% of all mRNA expression may be regulated by miRNAs, and some are either oncogenic or tumor suppressive. Complexity of miRNA regulation highlights the need for integrated assays, providing direct correlation between miRNA and mRNA, and protein expression. From a single 4 µm FFPE section, MultiOmyxTM hyperplexed immunofluorescent assay (demonstrated to stain up to 60 protein biomarkers) is utilized to measure CD3, CD4, CD8, CD16, CD56, Granzyme B, FoxP3, ICOS, OX40, OX40L, PD1, PDL1, HLA-DR, and Ki67 protein expression. From an adjacent 10 µm section, NanoStringTM nCounter PanCancer Immune Profiling Panel and Human v3 miRNA expression panel were utilized to comprehensively profile the expression of 770 mRNA and 800 miRNA.
Integrating MultiOmyx and NanoString technologies, the current study measured miRNA, mRNA, and protein expression in lung, head and neck, breast, and melanoma samples. For each indication, three samples were selected from a larger sample set, based on high protein expression of lymphocytes and macrophage markers (CD3, CD4, CD8, CD56, CD16), co-stimulator markers (ICOS, OX40), and immune checkpoint markers (PD1, PD-L1). Protein expression results indicate positive correlation between expression of ICOS and OX40 with higher infiltration of Thelper (CD3+CD4+), Tcytotoxic (CD3+CD8+), and effector T cells (CD3+CD8+Granzyme B). NanoString normalized mRNA counts for the protein biomarkers profiled indicate that all markers except for HLA-DR belong to low expressers group with counts ranging from 20-700. Comparison of protein expression to mRNA counts revealed inconsistencies in modulated markers (PD1, PD-L1, ICOS, OX40) which are attributed to differences in population of cells between the two sections. Direct assessment of up regulation of miRNA and down regulation of target mRNA could not be made for miRNAs reported in literature as negative regulator of PD-L1 (miR-34a, 34b, 34c), PD1 (miR-28, miR-107), FoxP3 (miR-210, miR-24, miR-31), and ICOS (miR-101). Analysis of multiple miRNAs (combinatorial targeting) mapped in context to mRNA, and their respective protein expression will be presented from lung, head and neck, breast, and melanoma cancer samples.
Citation Format: Qingyan Au, RaghavKrishna Padmanabhan, Nam Tran, Lakshmi Chandramohan, Nicholas Hoe. Integrated analysis of microRNA, mRNA, and protein expression utilizing MultiOmyxTM and NanoStringTM from formalin-fixed paraffin-embedded, lung, head and neck, breast, and melanoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1469. doi:10.1158/1538-7445.AM2017-1469