RNA Sequencing in Comparison to Immunohistochemistry for Measuring Cancer Biomarkers in Breast Cancer and Lung Cancer Specimens

Abstract

RNA sequencing is considered the gold standard for high-throughput profiling of gene expression at the transcriptional level. Its increasing importance in cancer research and molecular diagnostics is reflected in the growing number of its mentions in scientific literature and clinical trial reports. However, the use of different reagents and protocols for RNA sequencing often produces incompatible results. Recently, we published the Oncobox Atlas of RNA sequencing profiles for normal human tissues obtained from healthy donors killed in road accidents. This is a database of molecular profiles obtained using uniform protocol and reagents settings that can be broadly used in biomedicine for data normalization in pathology, including cancer. Here, we publish new original 39 breast cancer (BC) and 19 lung cancer (LC) RNA sequencing profiles obtained for formalin-fixed paraffin-embedded (FFPE) tissue samples, fully compatible with the Oncobox Atlas. We performed the first correlation study of RNA sequencing and immunohistochemistry-measured expression profiles for the clinically actionable biomarker genes in FFPE cancer tissue samples. We demonstrated high (Spearman’s rho 0.65–0.798) and statistically significant (p < 0.00004) correlations between the RNA sequencing (Oncobox protocol) and immunohistochemical measurements for HER2/ERBB2, ER/ESR1 and PGR genes in BC, and for PDL1 gene in LC; AUC: 0.963 for HER2, 0.921 for ESR1, 0.912 for PGR, and 0.922 for PDL1. To our knowledge, this is the first validation that total RNA sequencing of archived FFPE materials provides a reliable estimation of marker protein levels. These results show that in the future, RNA sequencing can complement immunohistochemistry for reliable measurements of the expression biomarkers in FFPE cancer samples.

ACE2 & TMPRSS2 Expressions in Head & Neck Tissues: A Systematic Review

“…Some studies interrogate gene expression at mRNA level and others at protein levels, both types of analysis having their advantages and limitations. Compared to transcriptomic analyzes, immunohistochemistry brings additional important spatial information in tissue samples but recently Sorokin et al demonstrated high and statistically significant correlations between the RNA sequencing and immunohistochemical measurements [42]. Interestingly, they highlighted the complementarity of both techniques for measuring cancer biomarkers in FFPE samples.…”

RNA Sequencing Data for FFPE Tumor Blocks Can Be Used for Robust Estimation of Tumor Mutation Burden in Individual Biosamples

“…Gene expression profiling by RNAseq is the alternative type of high-throughput genetic analysis that can inform clinical decision-making ( 51 , 52 ). It was recently published that RNAseq data can serve as the alternative to immunohistochemical tests for several major cancer markers like HER2, ESR1, PGR, and PD-L1 ( 22 ). It can reliably estimate concentrations of cancer drug targets ( 53 ), which is also true for the emerging non-protein molecular target ganglioside GD2 ( 27 ).…”

“…Thus, sequencing profiles derived from FFPE should be processed differently and the results obtained from fresh tissues may be poorly compatible. However, FFPE materials can be used to properly estimate TMB by the DNA screens ( 20 ) and for the clinical-grade estimation of the gene expression levels by analyzing RNA reads ( 21 , 22 ). From certain points of view, this is the preferred type of biomaterial because of its high availability and stability, as FFPE blocks can be stored at room temperature for years prior to nucleic acid extraction for sequencing purposes ( 23 ).…”

RNA Sequencing for Personalized Treatment of Metastatic Leiomyosarcoma: Case Report

“…RNA was isolated from FFPE slices and sequenced according to our previous protocols (20,60). DNA was extracted and used for whole-exome sequencing as described (30).…”