Evaluation of a Standardized Protocol for Processing Adrenal Tumor Samples: Preparation for a European Adrenal Tumor Bank

I. K. Johnsen; S. Hahner; J.-J. Brière; A. Ozimek; A. P. Gimenez-Roqueplo; C. Hantel; P. Adam; J. Bertherat; F. Beuschlein

doi:10.1055/s-0029-1241858

Hormone and Metabolic Research, Table of Contents

Horm Metab Res 2010; 42(2): 93-101
DOI: 10.1055/s-0029-1241858

Original Basic

Evaluation of a Standardized Protocol for Processing Adrenal Tumor Samples: Preparation for a European Adrenal Tumor Bank

I. K. Johnsen¹ , S. Hahner² , J.-J. Brière³ ^, ⁴ , A. Ozimek⁵ , A. P. Gimenez-Roqueplo⁶ ^, ⁷ ^, ⁸ , C. Hantel¹ , P. Adam⁹ , J. Bertherat⁷ ^, ¹⁰ ^, ¹¹ , F. Beuschlein¹ on Behalf of the European Network for the Study of Adrenal Tumors(ENS@T)*

¹Department of Medicine, University Hospital Innenstadt, Ludwig Maximilians University, Munich, Germany
²Department of Medicine, Endocrine and Diabetes Unit, Julius-Maximilian University, Würzburg, Germany
³INSERM, U676, Paris, France
⁴Université Paris 7, Faculté de Médecine Denis Diderot, IFR02, Paris, France
⁵Department of Surgery, University Hospital Innenstadt, Ludwig Maximilians University, Munich, Germany
⁶Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Genetics, Paris, France
⁷Université Paris-Descartes, Faculté de Médecine, Paris, France
⁸INSERM, U772, Collège de France, Paris, France
⁹Institute of Pathology, Julius-Maximilian University, Würzburg, Germany
¹⁰INSERM, U567, Institut Cochin, CNRS UMR 8104, Paris, France
¹¹Assistance Publique-Hôpitaux de Paris, Hôpital Cochin, Center for Rare Adrenal Diseases, Paris, France

Abstract

Storage and tissue handling of surgical tumor specimen have been recognized as critical steps that can potentially affect reproducibility and comparability of molecular endpoints between laboratories. In the preparation of adrenal tumor tissue banking, three different protocols that simulate warm ischemia upon tumor removal (protocol I), thawing and refreezing cycles (protocol II), as well as storage of vital tumor samples (protocol III) were applied. For the first two protocols, samples were subdivided and either snap frozen or treated with a RNA preserving agent (RPA) while in protocol III different storage media were compared. Following these procedures, recovery and integrity of DNA, RNA, and protein by means of pulsed field electrophoresis, long-range PCR, real-time PCR, immunoblot, and immunohistochemistry (protocol I and II) as well as cell viability and steroidogenic capacity (protocol III) were investigated. While DNA integrity was not influenced by different treatment modalities, expression levels of adrenal marker genes were more affected in samples after snap freezing in comparison to RPA pretreatment. Moreover, storage at room temperature before and after freezing could be demonstrated to decrease the relative amount of protein phosphorylation (ERK) and enzymatic activity (succinate cytochrome c reductase) while overall protein levels were not significantly affected. Similarly, morphological or immunohistochemical evaluation was comparable between groups. For primary cell cultures generated after storage of tumor samples similar rates of viability were observable while steroid output varied between the groups. Overall, on the basis of the presented endpoints standardized operational procedures can be defined for a proposed European adrenal tumor biobank.

Key words

adrenal tumor - tissue storage - tissue processing - standard operation procedure - molecular profiling - biobanking

Full Text

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1 See Acknowledgements for a listing of the ENS@T steering committee members.

Correspondence

F. BeuschleinMD

Department of Medicine, Endocrine Research University Hospital Innenstadt

Ziemssenstraße 1

80336 Munich

Germany

Phone: +49/89/5160 2110 (2116)

Fax: +49/89/5160 4467

Email: felix.beuschlein@med.uni-muenchen.de