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Updated: Dec 12, 2025
Comparative Lesions Analysis Through a Targeted Sequencing Approach
Published on: November 5, 2019
Targeted RNAseq of Formalin-Fixed Paraffin-Embedded Tissue to Differentiate Among Benign and Malignant Adrenal
Samuel W Plaska1, Chia-Jen Liu2,3, Jung Soo Lim1,4
1Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
Abstract:
Lack of routine fresh or frozen tissue is a barrier to widespread transcriptomic analysis of adrenal cortical tumors and an impediment to translational research in endocrinology and endocrine oncology. Our group has previously pioneered the use of targeted amplicon-based next-generation sequencing for archival formalin-fixed paraffin-embedded (FFPE) adrenal tissue specimens to characterize the spectrum of somatic mutations in various forms of primary aldosteronism. Herein, we developed and validated a novel 194-amplicon targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using clinical-grade FFPE specimens. Targeted RNAseq-derived expression values for 27 adrenal cortical tumors, including aldosterone-producing adenomas (APA; n=8), cortisol-producing adenomas (CPA; n=11), and adrenal cortical carcinomas (ACC; n=8), highlighted known differentially-expressed genes (DEGs; i. e., CYP11B2, IGF2, etc.) and tumor type-specific transcriptional modules (i. e., high cell cycle/proliferation transcript expression in ACC, etc.), and a subset of DEGs was validated orthogonally using quantitative reverse transcription PCR (qRT-PCR). Finally, unsupervised hierarchical clustering using a subset of high-confidence DEGs revealed three discrete clusters representing APA, CPA, and ACC tumors with corresponding unique gene expression signatures, suggesting potential clinical utility for a transcriptomic-based approach to tumor classification. Overall, these data support the use of targeted amplicon-based RNAseq for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material and indicate that this approach may facilitate important translational research opportunities for the study of these tumors.
Insights
This study introduces a targeted RNA sequencing assay for analyzing formalin-fixed paraffin-embedded adrenal tumors. The method successfully classifies adrenal tumors based on gene expression, aiding endocrine oncology research.
Area of Science:
- Endocrinology and Endocrine Oncology
- Genomics and Transcriptomics
- Molecular Pathology
Background:
- Widespread transcriptomic analysis of adrenal cortical tumors is hindered by the lack of routine fresh or frozen tissue.
- Archival formalin-fixed paraffin-embedded (FFPE) tissue is often the only available resource for studying adrenal tumors.
- Previous work demonstrated the utility of targeted next-generation sequencing for FFPE adrenal tissue mutation analysis.
Purpose of the Study:
- To develop and validate a targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using FFPE specimens.
- To assess the potential of this assay for classifying different types of adrenal cortical tumors.
Main Methods:
- Development and validation of a 194-amplicon targeted RNAseq assay for FFPE adrenal tissue.
- Transcriptomic profiling of 27 adrenal cortical tumors (8 APA, 11 CPA, 8 ACC).
- Orthogonal validation of differentially expressed genes (DEGs) using quantitative reverse transcription PCR (qRT-PCR).
- Unsupervised hierarchical clustering of gene expression data for tumor classification.
Main Results:
- The targeted RNAseq assay successfully identified known differentially-expressed genes (e.g., CYP11B2, IGF2) and tumor-specific transcriptional modules.
- High cell cycle and proliferation transcript expression was observed in adrenal cortical carcinomas (ACC).
- Unsupervised clustering revealed three distinct molecular subtypes corresponding to aldosterone-producing adenomas (APA), cortisol-producing adenomas (CPA), and ACC.
- Validated a subset of DEGs using qRT-PCR.
Conclusions:
- Targeted amplicon-based RNAseq is a viable method for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material.
- This approach demonstrates potential clinical utility for transcriptomic-based classification of adrenal tumors.
- The assay facilitates translational research opportunities in endocrinology and endocrine oncology using FFPE samples.
