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


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1


  1. 1.

    Dahia PL. Pheochromocytoma and paraganglioma pathogenesis: learning from genetic heterogeneity. Nat Rev Cancer. 2014;14:108–19.

  2. 2.

    Favier J, Amar L, Gimenez-Roqueplo AP. Paraganglioma and phaeochromocytoma: from genetics to personalized medicine. Nat Rev Endocrinol. 2015;11:101–11.

  3. 3.

    Remacha L, Comino-Mendez I, Richter S, Contreras L, Curras-Freixes M, Pita G, et al. Targeted exome sequencing of Krebs cycle genes reveals candidate cancer-predisposing mutations in pheochromocytomas and paragangliomas. Clin Cancer Res. 2017;2:6315–24.

  4. 4.

    Calsina B, Curras-Freixes M, Buffet A, Pons T, Contreras L, Leton R, et al. Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients. Genet Med. 2018;20:1652–62.

  5. 5.

    Remacha L, Curras-Freixes M, Torres-Ruiz R, Schiavi F, Torres-Perez R, Calsina B, et al. Gain-of-function mutations in DNMT3A in patients with paraganglioma. Genet Med. 2018;20:1644–51.

  6. 6.

    Buffet A, Morin A, Castro-Vega LJ, Habarou F, Lussey-Lepoutre C, Letouze E, et al. Germline mutations in the mitochondrial 2-oxoglutarate/malate carrier SLC25A11 gene confer a predisposition to metastatic paragangliomas. Cancer Res. 2018;78:1914–22.

  7. 7.

    Castro-Vega LJ, Lepoutre-Lussey C, Gimenez-Roqueplo AP, Favier J. Rethinking pheochromocytomas and paragangliomas from a genomic perspective. Oncogene. 2016;35:1080–9.

  8. 8.

    Toledo RA, Burnichon N, Cascon A, Benn DE, Bayley JP, Welander J, et al. Consensus statement on next-generation-sequencing-based diagnostic testing of hereditary phaeochromocytomas and paragangliomas. Nat Rev Endocrinol. 2017;13:233–47.

  9. 9.

    Ayala-Ramirez M, Chougnet CN, Habra MA, Palmer JL, Leboulleux S, Cabanillas ME, et al. Treatment with sunitinib for patients with progressive metastatic pheochromocytomas and sympathetic paragangliomas. J Clin Endocrinol Metab. 2012;97:4040–50.

  10. 10.

    Jimenez C, Cabanillas ME, Santarpia L, Jonasch E, Kyle KL, Lano EA, et al. Use of the tyrosine kinase inhibitor sunitinib in a patient with von Hippel-Lindau disease: targeting angiogenic factors in pheochromocytoma and other von Hippel-Lindau disease-related tumors. J Clin Endocrinol Metab. 2009;94:386–91.

  11. 11.

    van Nederveen FH, Gaal J, Favier J, Korpershoek E, Oldenburg RA, de Bruyn EM, et al. An immunohistochemical procedure to detect patients with paraganglioma and phaeochromocytoma with germline SDHB, SDHC, or SDHD gene mutations: a retrospective and prospective analysis. Lancet Oncol. 2009;10:764–71.

  12. 12.

    Burnichon N, Briere JJ, Libe R, Vescovo L, Riviere J, Tissier F, et al. SDHA is a tumor suppressor gene causing paraganglioma. Hum Mol Genet. 2010;19:3011–20.

  13. 13.

    Korpershoek E, Favier J, Gaal J, Burnichon N, van Gessel B, Oudijk L, et al. SDHA immunohistochemistry detects germline SDHA gene mutations in apparently sporadic paragangliomas and pheochromocytomas. J Clin Endocrinol Metab. 2011;96:E1472–6.

  14. 14.

    Menara M, Oudijk L, Badoual C, Bertherat J, Lepoutre-Lussey C, Amar L, et al. SDHD immunohistochemistry: a new tool to validate SDHx mutations in pheochromocytoma/paraganglioma. J Clin Endocrinol Metab. 2015;100:E287–91.

  15. 15.

    Papathomas TG, Oudijk L, Persu A, Gill AJ, van Nederveen F, Tischler AS, et al. SDHB/SDHA immunohistochemistry in pheochromocytomas and paragangliomas: a multicenter interobserver variation analysis using virtual microscopy: a Multinational Study of the European Network for the Study of Adrenal Tumors (ENS@T). Mod Pathol. 2015;28:807–21.

  16. 16.

    Castro-Vega LJ, Buffet A, De Cubas AA, Cascon A, Menara M, Khalifa E, et al. Germline mutations in FH confer predisposition to malignant pheochromocytomas and paragangliomas. Hum Mol Genet. 2014;23:2440–6.

  17. 17.

    Burnichon N, Cascon A, Schiavi F, Morales NP, Comino-Mendez I, Abermil N, et al. MAX mutations cause hereditary and sporadic pheochromocytoma and paraganglioma. Clin Cancer Res. 2012;18:2828–37.

  18. 18.

    Vogel TW, Vortmeyer AO, Lubensky IA, Lee YS, Furuta M, Ikejiri B, et al. Coexpression of erythropoietin and its receptor in endolymphatic sac tumors. J Neurosurg. 2005;103:284–8.

  19. 19.

    Nakamura E, Abreu-e-Lima P, Awakura Y, Inoue T, Kamoto T, Ogawa O, et al. Clusterin is a secreted marker for a hypoxia-inducible factor-independent function of the von Hippel-Lindau tumor suppressor protein. Am J Pathol. 2006;168:574–84.

  20. 20.

    Pinato DJ, Ramachandran R, Toussi ST, Vergine M, Ngo N, Sharma R, et al. Immunohistochemical markers of the hypoxic response can identify malignancy in phaeochromocytomas and paragangliomas and optimize the detection of tumours with VHL germline mutations. Br J Cancer. 2013;108:429–37.

  21. 21.

    Al-Ahmadie HA, Alden D, Qin LX, Olgac S, Fine SW, Gopalan A, et al. Carbonic anhydrase IX expression in clear cell renal cell carcinoma: an immunohistochemical study comparing 2 antibodies. Am J Surg Pathol. 2008;32:377–82.

  22. 22.

    Burnichon N, Vescovo L, Amar L, Libe R, de Reynies A, Venisse A, et al. Integrative genomic analysis reveals somatic mutations in pheochromocytoma and paraganglioma. Hum Mol Genet. 2011;20:3974–85.

  23. 23.

    Castro-Vega LJ, Letouze E, Burnichon N, Buffet A, Disderot PH, Khalifa E, et al. Multi-omics analysis defines core genomic alterations in pheochromocytomas and paragangliomas. Nat Commun. 2015;6:6044.

  24. 24.

    Ben Aim L, Pigny P, Castro-Vega LJ, Buffet A, Amar L, Bertherat J, et al. Targeted next-generation sequencing detects rare genetic events in pheochromocytoma and paraganglioma. J Med Genet. 2019;56:513–20.

  25. 25.

    Yates CJ, McAuley SA, Grodski S, Hamblin PS, Ebeling PR. An elusive phaeochromocytoma. Med J Aust. 2011;194:44–5.

  26. 26.

    Nordstrom-O’Brien M, van der Luijt RB, van Rooijen E, van den Ouweland AM, Majoor-Krakauer DF, Lolkema MP, et al. Genetic analysis of von Hippel-Lindau disease. Hum Mutat 2010;31:521–37.

  27. 27.

    Lenglet M, Robriquet F, Schwarz K, Camps C, Couturier A, Hoogewijs D, et al. Identification of a new VHL exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease. Blood. 2018;132:469–83.

  28. 28.

    Lenders JW, Duh QY, Eisenhofer G, Gimenez-Roqueplo AP, Grebe SK, Murad MH, et al. Pheochromocytoma and paraganglioma: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2014;99:1915–42.

  29. 29.

    Buffet A, Ben Aim L, Leboulleux S, Drui D, Vezzosi D, Libe R, et al. Positive impact of genetic test on the management and outcome of patients with paraganglioma and/or pheochromocytoma. J Clin Endocrinol Metab. 2019;104:1109–18.

  30. 30.

    Curras-Freixes M, Pineiro-Yanez E, Montero-Conde C, Apellaniz-Ruiz M, Calsina B, Mancikova V, Remacha L, et al. PheoSeq: a targeted next-generation sequencing assay for pheochromocytoma and paraganglioma diagnostics. J Mol Diagn. 2017;19:575–88.

  31. 31.

    Toledo RA, Dahia PL. Next-generation sequencing for the diagnosis of hereditary pheochromocytoma and paraganglioma syndromes. Curr Opin Endocrinol Diabetes Obes. 2015;22:169–79.

  32. 32.

    Vanderveen KA, Thompson SM, Callstrom MR, Young WF Jr., Grant CS, Farley DR, et al. Biopsy of pheochromocytomas and paragangliomas: potential for disaster. Surgery. 2009;146:1158–66.

Download references


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).


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).

Author information

Correspondence to Judith Favier.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.


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 ( Identifier: NCT02672020).

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Figure S1

Supplemental Table 1

Supplementary Table 2

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark