Global DNA Methylation Analysis Identifies Two Discrete clusters of Pheochromocytoma with Distinct Genomic and Genetic Alterations

Samuel Backman, Rajani Maharjan, Alberto Falk-Delgado, Joakim Crona, Kenko Cupisti, Peter Stålberg, Per Hellman, Peyman Björklund, Samuel Backman, Rajani Maharjan, Alberto Falk-Delgado, Joakim Crona, Kenko Cupisti, Peter Stålberg, Per Hellman, Peyman Björklund

Abstract

Pheochromocytomas and paragangliomas (PPGLs) are rare and frequently heritable neural-crest derived tumours arising from the adrenal medulla or extra-adrenal chromaffin cells respectively. The majority of PPGL tumours are benign and do not recur with distant metastases. However, a sizeable fraction of these tumours secrete vasoactive catecholamines into the circulation causing a variety of symptoms including hypertension, palpitations and diaphoresis. The genetic landscape of PPGL has been well characterized and more than a dozen genes have been described as recurrently mutated. Recent studies of DNA-methylation have revealed distinct clusters of PPGL that share DNA methylation patterns and driver mutations, as well as identified potential biomarkers for malignancy. However, these findings have not been adequately validated in independent cohorts. In this study we use an array-based genome-wide approach to study the methylome of 39 PPGL and 4 normal adrenal medullae. We identified two distinct clusters of tumours characterized by different methylation patterns and different driver mutations. Moreover, we identify genes that are differentially methylated between tumour subcategories, and between tumours and normal tissue.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Heatmap and hierarchical clustering of…
Figure 1. Heatmap and hierarchical clustering of tumour samples based on DNA methylation.
Two distinct clusters are observed. The symbols *, #, and § denote samples originating from the same cases.
Figure 2
Figure 2
(a) Principal components analysis of the tumour samples shows the two clusters separated along the first principal component. (b) Methylation index of tumours in the two clusters, and of normal adrenal medulla. Tumours in cluster A have a lower global methylation level than tumours in cluster B and normal tissue. (c) Methylation index of benign tumours, malignant tumours and normal tissue.
Figure 3
Figure 3
(a) Venn diagram of probes aberrantly methylated in the two clusters (compared to normal tissue). (b) Venn diagram of probes aberrantly methylated in benign and malignant tumours, compared to normal tissue.
Figure 4
Figure 4
(a) Number of Somatic Copy Number Aberrations (SCNAs) in the two clusters. Tumours in cluster B carry more SCNAs than tumours in cluster A. (b) Number of SCNAs in malignant and benign tumours. Malignant tumours carry more SCNAs.
Figure 5
Figure 5
(a) PNMT mRNA expression is decreased in tumours with high levels of PNMT promoter methylation. (b) Tumours in cluster B tend to have higher levels of PNMT promoter methylation than tumours in cluster A.
Figure 6
Figure 6
(a) Receiver Operating Characteristic (ROC) curve for a CpG probe associated with RDBP previously suggested as a biomarker of metastatic disease. (b) ROC curve for the other probes associated with RDBP on array.

References

    1. Stenström G. & Svärdsudd K. Pheochromocytoma in Sweden 1958–1981. An analysis of the National Cancer Registry Data. Acta Med. Scand. 3, 225–232 (1986).
    1. Beard C., Sheps S., Kurland L., Carney J. & Lie J. Occurence of pheochromocytoma in Rochester, Minnesota, 1950 through 1979. Mayo Clin. Proc. 12, 802–804 (1983).
    1. Favier J., Amar L. & Gimenez-Roqueplo A.-P. Paraganglioma and phaeochromocytoma: from genetics to personalized medicine. Nature Reviews Endocrinology 11, 101–111 (2015).
    1. Zhuang Z. et al.. Somatic HIF2A Gain-of-Function Mutations in Paraganglioma with Polycythemia. New England Journal of Medicine 367, 922–930 (2012).
    1. Qin Y. et al.. Germline mutations in TMEM127 confer susceptibility to pheochromocytoma. Nature genetics 42, 229–233 (2010).
    1. Comino-Méndez I. et al.. Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma. Nature genetics 43, 663–667 (2011).
    1. Burnichon N. et al.. Somatic NF1 inactivation is a frequent event in sporadic pheochromocytoma. Human Molecular Genetics 21, 5397–5405 (2012).
    1. Clark G. et al.. Germline FH Mutations Presenting With Pheochromocytoma. Journal of Clinical Endocrinology and Metabolism 99, E2046–E2050 (2014).
    1. Burnichon N. et al.. SDHA is a tumor suppressor gene causing paraganglioma. Human molecular genetics 19, 3011–3020, doi: 10.1093/hmg/ddq206 (2010).
    1. Astuti D. et al.. Gene mutations in the succinate dehydrogenase subunit SDHB cause susceptibility to familial pheochromocytoma and to familial paraganglioma. American journal of human genetics 69, 49–54, doi: 10.1086/321282 (2001).
    1. Niemann S. & Muller U. Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nature genetics 26, 268–270, doi: 10.1038/81551 (2000).
    1. Baysal B. E. et al.. Mutations in SDHD, a mitochondrial complex II gene, in hereditary paraganglioma. Science 287, 848–851 (2000).
    1. Bayley J. P. et al.. SDHAF2 mutations in familial and sporadic paraganglioma and phaeochromocytoma. The Lancet. Oncology 11, 366–372, doi: 10.1016/S1470-2045(10)70007-3 (2010).
    1. Cascon A. et al.. Whole-exome sequencing identifies MDH2 as a new familial paraganglioma gene. J Natl Cancer Inst. 107 (2015).
    1. Crona J. et al.. Somatic mutations in H-RAS in sporadic pheochromocytoma and paraganglioma identified by exome sequencing. Journal of Clinical Endocrinology and Metabolism 7, 1266–1271 (2013).
    1. Juhlin C. C. et al.. Whole-exome sequencing defines the mutational landscape of pheochromocytoma and identifies KMT2D as a recurrently mutated gene. Genes, chromosomes & cancer 54, 542–554, doi: 10.1002/gcc.22267 (2015).
    1. Fishbein L. et al.. Whole-exome sequencing identifies somatic ATRX mutations in pheochromocytomas and paragangliomas. Nature communications 6, 6140, doi: 10.1038/ncomms7140 (2015).
    1. Burnichon N. et al.. Integrative genomic analysis reveals somatic mutations in pheochromocytoma and paraganglioma. Human Molecular Genetics 20, 3974–3985 (2011).
    1. Kim W. & Kaelin W. Role of VHL Gene Mutation in Human Cancer. Journal of Clinical Oncology 22, 4991–5004 (2004).
    1. Selak M. et al.. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-α prolyl hydroxylase. Cancer Cell 7, 77–85 (2005).
    1. Dahlia P. L. M. Pheochromocytoma and paraganglioma pathogenesis: learning from genetic heterogeneity. Nature Reviews Cancer 14, 108–119 (2014).
    1. Amar L. et al.. Succinate Dehydrogenase B Gene Mutations Predict Survival in Patients with Malignant Pheochromocytomas or Paragangliomas. Journal of Clinical Endocrinology and Metabolism 92 (2007).
    1. Jones P. & Baylin S. The Epigenomics of Cancer. Cell 128, 683–692 (2007).
    1. *Barreau O. et al.. Identification of CpG Island Methylator Phenotype in Adrenocortical Carcinomas. Journal of Clinical Endocrinology and Metabolism 98, E174–E184 (2012).
    1. Toyota M. et al.. CpG island methylator phenotype in colorectal cancer. Proceedings of the National Academy of Sciences 96, 8681–8686 (1999).
    1. Noushmehr H. et al.. Identification of CpG Island Methylator Phenotype that Defines a Distinct Subgroup of Glioma. Cancer Cell 17, 510–522 (2010).
    1. Hegi M. et al.. MGMT Gene Silencing and Benefit from Temozolomide in Glioblastoma. New England Journal of Medicine 352, 997–1003 (2005).
    1. Geli J. et al.. The Ras effectors NORE1A and RASSF1A are frequently inactivated in pheochromocytoma and abdominal paraganglioma. Endocrine-related cancer 14, 125–134 (2007).
    1. Muscarella P. et al.. Expression of the p16INK4A/Cdkn2a gene is prevalently downregulated in human pheochromocytoma tumor specimens. Gene Expr 14, 207–216 (2008).
    1. de Cubas A. A. et al.. DNA Methylation Profiling in Pheochromocytoma and Paraganglioma Reveals Diagnostic and Prognostic Markers. Clinical cancer research: an official journal of the American Association for Cancer Research 21, 3020–3030, doi: 10.1158/1078-0432.CCR-14-2804 (2015).
    1. Letouzé E. et al.. SDH mutations establish a hypermethylator phenotype in paraganglioma. Cancer Cell 23, 749–752 (2013).
    1. Wang J., Chen L., Li Y. & Guan X. Y. Overexpression of cathepsin Z contributes to tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma. PloS one 6, e24967, doi: 10.1371/journal.pone.0024967 (2011).
    1. Liu Q., Zhang C., Ma G. & Zhang Q. Expression of SPRR3 is associated with tumor cell proliferation and invasion in glioblastoma multiforme. Oncology Letters 7, 427–432 (2014).
    1. Cho D. et al.. Upregulation of SPRR3 promotes colorectal tumorigenesis. Molecular Medicine 16, 271–277 (2010).
    1. Brocks D. et al.. Intratumour DNA methylation Heterogeneity Reflects Clonal Evolution in Aggressive Prostate Cancer. Cell Repots 8, 798–806 (2014).
    1. Crona J. et al.. Spatiotemporal Heterogeneity Characterizes the Genetic Landscape of Pheochromocytoma and Defines Early Events in Tumorigenesis. Clinical cancer research: an official journal of the American Association for Cancer Research 21, 4451–4460, doi: 10.1158/1078-0432.CCR-14-2854 (2015).
    1. Crona J. et al.. Integrative genetic characterization and phenotype correlations in pheochromocytoma and paraganglioma tumours. PloS one 9, e86756, doi: 10.1371/journal.pone.0086756 (2014).
    1. Van Loo P. et al.. Allele-specific copy number analysis of tumors. Proceedings of the National Academy of Sciences of the United States of America 107, 16910–16915, doi: 10.1073/pnas.1009843107 (2010).
    1. Benjamini Y. & Hochberg Y. Controlling the False Discovery Rate - a Practical and Powerful Approach to Multiple Testing. J Roy Stat Soc B Met 57, 289–300 (1995).
    1. Klipper-Aurbach Y. et al.. Mathematical formulae for the prediction of the residual beta cell function during the first two years of disease in children and adolescents with insulin-dependent diabetes mellitus. Med Hypotheses 45, 486–490 (1995).
    1. Hochberg Y. & Benjamini Y. More powerful procedures for multiple significance testing. Stat Med 9, 811–818 (1990).

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