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Horm Metab Res 2020; 52(08): 607-613
DOI: 10.1055/a-1212-8803
Endocrine Research

Targeted RNAseq of Formalin-Fixed Paraffin-Embedded Tissue to Differentiate Among Benign and Malignant Adrenal Cortical Tumors

Samuel W. Plaska 
1   Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Chia-Jen Liu
2   Michigan Center for Translational Pathology, Ann Arbor, Michigan, USA
3   Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Jung Soo Lim
1   Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA
4   Division of Endocrinology and Metabolism, Department of Internal Medicine, Yonsei University Wonju College of Medicine, Wonju, South Korea
,
Juilee Rege
1   Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Nolan R. Bick
1   Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA
3   Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Antonio M. Lerario
5   Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Gary D. Hammer
1   Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA
5   Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan, USA
6   Department of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, USA
7   Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan, USA
,
Thomas J. Giordano
3   Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA
5   Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan, USA
7   Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan, USA
,
Tobias Else
5   Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
Scott A. Tomlins
3   Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA
,
William E. Rainey
1   Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, USA
5   Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical School, Ann Arbor, Michigan, USA
7   Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan, USA
,
Aaron M. Udager
2   Michigan Center for Translational Pathology, Ann Arbor, Michigan, USA
3   Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA
7   Rogel Cancer Center, University of Michigan, Ann Arbor, Michigan, USA
› Author Affiliations Funding Information W.E.R. is supported by the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK106618). G.D.H. is supported by the Department of Defense Congressionally Directed Medical Research Programs (W81XWH-19-1-0623 and W81XWH-19-1-0528).
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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.

Key words

next-generation sequencing (NGS) - quantitative reverse-transcription polymerase chain reaction (qRT-PCR) - aldosterone-producing adenoma (APA) - cortisol-producing adenoma (CPA) - adrenal cortical carcinoma (ACC) - FFPE

Supplementary Material



Publication History

Received: 12 May 2020

Accepted: 24 June 2020

Article published online:
13 August 2020

© Georg Thieme Verlag KG
Stuttgart · New York

 

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