Carbonic anhydrase 9 immunohistochemistry as a tool to predict or validate germline and somatic VHL mutations in pheochromocytoma and paraganglioma—a retrospective and prospective study

Abstract

The development of pheochromocytomas and paragangliomas is strongly linked to the presence of germline mutations in more than 15 predisposing genes. Among them, germline and somatic VHL mutations account for ~10% of all cases. In contrast with SDHA and SDHB immunohistochemistries that are routinely used to validate SDHx gene mutations, there is no such tool available for VHL mutations. The aim of this study was to evaluate whether CA9 immunostaining could be used as a tool to predict the presence or validate the pathogenicity of VHL gene mutations in paraganglioma. Immunohistochemistry for CA9 was performed on 207 tumors. A retrospective series of 100 paragangliomas with known mutation status for paraganglioma susceptibility genes was first investigated. Then, a prospective series of 107 paragangliomas was investigated for CA9 immunostaining followed by germline and/or somatic genetic testing of all paraganglioma susceptibility genes by next-generation sequencing. Cytosolic CA9 protein expression was heterogeneous in the different samples. However, we observed that a membranous CA9 staining was almost exclusively observed in VHL-related cases. Forty two of 48 (88%) VHL-mutated samples showed a CA9 membranous immunostaining. Positive cells were either isolated, varying from 1 or 2 cells (5% of cases) to 10–20 cells per tumor block (35% of cases), grouped in areas of focal positivity representing between 1 and 20% of the tissue section (35% of cases), or widely distributed on 80–100% of the tumor sections (25% of samples). In contrast, 142/159 (91%) of non-VHL-mutated tumors presented no membrane CA9 localization. Our results demonstrate that VHL gene mutations can be predicted or validated reliably by an easy-to-perform and low-cost immunohistochemical procedure. CA9 immunohistochemistry on paragangliomas will improve the diagnosis of VHL-related disease, which is important for the surveillance and therapeutic management of paraganglioma patients, and in case of germline mutation, their family members.

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Acknowledgements

We are grateful to Caroline Travers, Valentin Adamus, Nelly Le Pottier, Mathieu Madelaine, Karine Auribault, Patrick Nitschke, and Annabelle Venisse for their technical contribution to this work as well as Catherine Tritscher for administrative assistance. We thank the Biological Resources Center and Tumor Bank Platform, Hôpital Européen Georges Pompidou (BB-0033-00063). We are specifically grateful to the following pathologists, investigators of the COMETE-TACTIC consortium, who provided tumor tissues as well as their local biological resources centers: Dr Abir Ghuzlan (Gustave Roussy, Villejuif), Dr Frédérique Tissier (AP-HP, Hôpital de la Pitié Salpétrière, Paris), Dr Sophie Michalak (CHU Angers), Dr Valérie Coste-Martineau (Hôpital Arnaud de Villeneuve, CHU Montpellier), Dr Yves Allory (AP-HP, Hôpital Henri Mondor, Créteil), Pr Catherine Guettier-Bouttier (AP-HP, Hôpital de Bicêtre, Le Kremlin-Bicêtre), Pr Jean-Philippe Merlio (CHU Bordeaux), Dr Karine Renaudin (CHU Nantes), Pr Nathalie Sturm (CHU Grenoble), Dr Myriam Decaussin-Petrucci (CHU Lyon), Dr Serge Guyetant (CHU Tours), Dr Danielle Diebold (CHU de Reims), Pr Nathalie Rioux-Leclercq (CHU Rennes), Pr Marie-Pierre Neu-Chenard (CHU Strasbourg), and Pr Anne Gomez-Brouchet (CHU Toulouse).

Funding

This work has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no 633983 and by the Institut National du Cancer and the Direction Générale de l’Offre de Soins (PRT-K 2014, COMETE-TACTIC, INCa-DGOS_8663). NB received a financial support from the Cancer Research for Personalized Medicine—CARPEM project (Site de Recherche Intégré sur le Cancer—SIRIC). Our team is supported by the Ligue Nationale contre le Cancer (Equipe Labellisée).

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Correspondence to Judith Favier.

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The authors declare that they have no conflict of interest.

Ethics

All patients provided written informed consent for paraganglioma genetic testing, collection of samples, and subsequent analyses. Ethical approval for the study was obtained from the institutional review board (IRB 00003835, Comité de Protection des Personnes Ile de France IV, September 2015). This work is a part of the COMETE-TACTIC study (ClinicalTrials.gov Identifier: NCT02672020).

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