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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

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Robust detection of immune transcripts in FFPE samples using targeted RNA sequencing.

Benjamin E Paluch¹, Sean T Glenn^1,2, Jeffrey M Conroy^1,3

¹Omniseq LLC, Buffalo, NY 14203, USA.

|December 3, 2016

Summary

A new RNA sequencing (RNAseq) method accurately measures immune cell markers in preserved cancer tissues. This approach improves patient selection for immunotherapy, overcoming limitations of current subjective methods.

Keywords:

CD8+ cytotoxic T lymphocytes NY-ESO-1 PD-L1 cancer immunotherapy nivolumab

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Published on: May 5, 2018

Area of Science:

Oncology
Molecular Biology
Immunology

Background:

Current methods for selecting cancer patients for immune checkpoint blockers (ICBs) rely on subjective assessment of PD-L1 protein levels via immunohistochemistry (IHC), which can be misinterpreted.
There is a need for objective and sensitive methods to identify patients who will benefit from immunotherapy.

Purpose of the Study:

To develop and validate a targeted RNA sequencing (RNAseq) approach for quantifying immune transcript abundance in formalin-fixed paraffin-embedded (FFPE) cancer specimens.
To assess the reliability and concordance of this RNAseq method with existing techniques and sample types.

Main Methods:

A targeted RNAseq panel was designed to measure immune transcript expression.
The assay was applied to FFPE ovarian carcinoma samples.
RNAseq results were compared between FFPE and freshly frozen (FF) samples from the same tumors.
RNAseq data was correlated with quantitative RT-PCR (qRT-PCR) and IHC protein expression levels.

Main Results:

The targeted RNAseq panel reliably profiled mRNA expression in FFPE samples.
Immune transcript expression measured by RNAseq in FFPE samples showed high concordance with FF samples, despite RNA degradation.
Targeted RNAseq results on FFPE specimens strongly correlated with qRT-PCR mRNA levels and IHC protein abundance.

Conclusions:

Targeted RNAseq is a sensitive and reliable method for profiling immune transcripts in archival FFPE tissues.
This RNAseq approach offers an objective alternative to IHC for assessing biomarkers relevant to cancer immunotherapy.
RNAseq profiling of FFPE tissues can be effectively utilized in research evaluating cancer immunotherapy efficacy.