Virchows Archiv

, Volume 465, Issue 3, pp 321–330 | Cite as

Mutational status of VHL gene and its clinical importance in renal clear cell carcinoma

  • Mariana Rezende Alves
  • Felipe Cavalcanti Carneiro
  • André Mourão Lavorato-Rocha
  • Walter Henriques da Costa
  • Isabela Werneck da Cunha
  • Stênio de Cássio Zequi
  • Gustavo Cardoso Guimaraes
  • Fernando Augusto Soares
  • Dirce Maria Carraro
  • Rafael Malagoli Rocha
Original Article


The most common subtype of renal cell carcinoma is the clear cell type (ccRCC), accounting for 75 % of cases. Inactivation of VHL gene is thought to be an early event in ccRCC carcinogenesis. Our intention was to assess whether VHL mutational status might provide useful predictive or prognostic information in patients with ccRCC. VHL messenger RNA (mRNA) expression was analyzed by in situ hybridization and its protein by immunohistochemistry on a tissue microarray containing samples from 148 cases. This was validated by qRT-PCR on 62 cases, for which RNA was available. The mutation status was assessed in 91 cases by Sanger sequencing. VHL was found mutated in 57 % of cases, with missense mutations in 26 %, nonsense in 5 %, splice site in 13 %, deletions in 39 %, indels in 8 %, duplications in 8 %, and insertions in 2 % of the cases. The prevalence of mutations by exon was the following: exon 1, 47 %; exon 2, 27 %; and exon 3, 13 %. VHL protein was expressed in a high number of cases (80 %), but significant correlations were not found between protein expression, clinical data, and survival. Importantly, of the 91 samples evaluated by sequencing, 45 were mutated, and 87 % of those were strongly positive. We found 32 novel mutations in the VHL gene in ccRCC. The presence of mutations was not concordant with mRNA or protein expression. Nonsense mutations of the VHL gene appear to be related with poorer prognosis and survival.


Renal carcinoma Clear cell Prognosis Mutational status VHL 



Clear cel renal carcinoma


Cullin 2


Formalin-fixed paraffin-embedded


The Human Gene Mutation Database


Hypoxia-inducible factor 1-alpha




In situ hybridization


Ring-box protein 1


Renal cell carcinoma


Reverse transcription polymerase chain reaction


Department of Medical Records and Statistics


Classification of malignant Tumors


Von Hippel–Lindau

Supplementary material

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Supplementary Table 1(DOCX 21 kb)
428_2014_1629_MOESM2_ESM.docx (17 kb)
Supplementary Table 2(DOCX 16 kb)
428_2014_1629_MOESM3_ESM.docx (17 kb)
Supplementary Table 3(DOCX 17 kb)
428_2014_1629_MOESM4_ESM.docx (24 kb)
Supplementary Table 4(DOCX 24 kb)


  1. 1.
    Lopez-Beltran A, Carrasco JC, Cheng L et al (2009) Update on the classification of renal epithelial tumors in adults. Int J Urol 16:432–443. doi:10.1111/j.1442-2042.2009.02302 PubMedCrossRefGoogle Scholar
  2. 2.
    Kaelin WG Jr (2002) Molecular basis of the VHL hereditary cancer syndrome. Nat Rev Cancer 2(9):673–682. doi:10.1038/nrc885 PubMedCrossRefGoogle Scholar
  3. 3.
    Latif F, Tory K, Gnarra J et al (1993) Identification of the von Hippel-Lindau disease tumor suppressor gene. Science 260(5112):1317–1320PubMedCrossRefGoogle Scholar
  4. 4.
    Kamura T, Koepp DM, Conrad MN et al (1999) Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase. Science 284(5414):657–661PubMedCrossRefGoogle Scholar
  5. 5.
    Carew JS, Esquivel JA, Espitia CM et al (2012) ELR510444 inhibits tumor growth and angiogenesis by abrogating HIF activity and disrupting microtubules inrenal cell carcinoma. PLoS One 7(1):e31120. doi:10.1371/journal.pone.0031120 PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Gossage L, Eisen T (2010) Alterations in VHL as potential biomarkers in renal-cell carcinoma. Nat Rev Clin Oncol 7(5):277–288. doi:10.1038/nrclinonc.2010.42 PubMedCrossRefGoogle Scholar
  7. 7.
    Tyers M, Willems AR (1999) One ring to rule a superfamily of E3 ubiquitin ligases. Science 284(5414):601–604. doi:10.1126/science.284.5414.601 PubMedCrossRefGoogle Scholar
  8. 8.
    Kaelin WG Jr (2007) von Hippel-Lindau Disease. Annu Rev Pathol 2:145–173. doi:10.1146/annurev.pathol.2.010506.092049 PubMedCrossRefGoogle Scholar
  9. 9.
    Baldewijns MM, van Vlodrop IJ, Vermeulen PB et al (2010) VHL and HIF signalling in renal cell carcinogenesis. J Pathol 221(2):125–138. doi:10.1002/path.2689 PubMedCrossRefGoogle Scholar
  10. 10.
    Kim WY, Kaelin WG (2004) Role of VHL gene mutation in human cancer. J Clin Oncol 22(24):4991–5004. doi:10.1007/s13277-011-0257-3. Epub 2011 Nov 29 PubMedCrossRefGoogle Scholar
  11. 11.
    Pause A, Lee S, Lonergan KM et al (1998) The von Hippel-Lindau tumor suppressor gene is required for cell cycle exit upon serum withdrawal. Proc Natl Acad Sci U S A 95(3):993–998PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Ohh M, Park CW, Ivan M et al (2000) Ubiquitination of hypoxia-inducible factor requires direct binding to the beta-domain of the von Hippel-Lindau protein. Nat Cell Biol 2(7):423–427PubMedCrossRefGoogle Scholar
  13. 13.
    Nickerson ML, Jaeger E, Shi Y et al (2008) Improved identification of von Hippel-Lindau gene alterations in clear cell renal tumors. Clin Cancer Res 14(15):4726–4734. doi:10.1158/1078-0432.CCR-07-4921 PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Young AC, Craven RA, Cohen D, Taylor C, Booth C et al (2009) Analysis of VHL gene alterations and their relationship to clinical parameters in sporadic conventional renal cell carcinoma. Clin Cancer Res 15(24):7582–7592. doi:10.1158/1078-0432.CCR-09-2131 PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Fuhrman SA, Lasky LC, Limas C (1982) Prognostic significance of morphologic parameters in renal cell carcinoma. Am J Surg Pathol 6(7):655–663PubMedCrossRefGoogle Scholar
  16. 16.
    Rioux-Leclercq N, Turlin B, Bansard J et al (2000) Value of immunohistochemical Ki- 67 and p53 determinations as predictive factors of outcome in renal cellcarcinoma. Urology 5(4):501–505CrossRefGoogle Scholar
  17. 17.
    Edge SB, Byrd DR, Compton CC et al (2009) AJCC Cancer Staging Manual. Springer; 7th ed.XV, 649pGoogle Scholar
  18. 18.
    Rocha RM, Miller K, Soares F et al (2009) Biotin-free systems provide stronger immunohistochemical signal in oestrogen receptor evaluation of breast câncer. J Clin Pathol 62(8):699–704. doi:10.1136/jcp.2009.065326 PubMedCrossRefGoogle Scholar
  19. 19.
    McCarty KS Jr, Szabo E, Flowers JL et al (1986) Use of a monoclonal anti-strogen receptor antibody in the immunohistochemical evaluation of human tumors. Cancer Res 46(8 Suppl):4244s–4248sPubMedGoogle Scholar
  20. 20.
    Wang WC, Chen HJ, Tseng YH et al (2009) Identification of somatic mutations in the von Hippel-Lindau (VHL) gene in a patient with renal cell carcinoma. J Formos Med Assoc 108(11):886–893. doi:10.1016/S0929-6646(09)60421-6 PubMedCrossRefGoogle Scholar
  21. 21.
    Yao M, Yoshida M, Kishida T et al (2002) VHL tumor suppressor gene alteration associated with good prognosis in sporadic clear-cell renal carcinoma. J Natl Cancer Inst 94(20):1569–1575PubMedCrossRefGoogle Scholar
  22. 22.
    Kondo K, Yao M, Yoshida M, Kishida T et al (2002) Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: relationship to clinicopathological parameters. Genes Chromosomes Cancer 34(1):58–68PubMedCrossRefGoogle Scholar
  23. 23.
    Schraml P, Struckmann K, Hatz F et al (2002) VHL mutations and their correlation with tumors cell cell proliferation, microvessel density, and patient prognosis inclear cell renal cell carcinoma. J Pathol 196(2):186–193PubMedCrossRefGoogle Scholar
  24. 24.
    Rechsteiner MP, von Teichman A, Nowicka A et al (2011) VHL gene mutations and their effects on hypoxia inducible factor HIFα: identification of potential driver and passenger mutations. Cancer Res 71(16):5500–5511. doi:10.1158/0008-5472.CAN-11-0757 PubMedCrossRefGoogle Scholar
  25. 25.
    Banks RE, Tirukonda P, Taylor C et al (2006) Genetic and epigenetic analysis of von Hippel- Lindal (VHL) gene alteration and relation with clinical variables in sporadic renal cancer. Cancer Res 66(4):2000–2011PubMedCrossRefGoogle Scholar
  26. 26.
    Rocha R, Nunes C, Rocha G et al (2008) Rabbit monoclonal antibodies show higher sensitivity than mouse for estrogen and progesteronereceptor evaluation in breast cancer by immunohistochemistry. Pathol Res Pract 204(9):655–662. doi:10.1016/j.prp.2008.03.010 PubMedCrossRefGoogle Scholar
  27. 27.
    Patard JJ, Rioux-Leclercq N, Masson D et al (2009) Absence of VHL gene alteration and high VEGF expression are associated with tumour aggressiveness and poorsurvival of renal-cell carcinoma. Br J Cancer 101(8):1417–1424. doi:10.1038/sj.bjc.6605298 PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Thusberg J, Vihinen M (2009) Pathogenic or not? And if so, then how? Studying the effects of missense mutations using bioinformatics methods. Hum Mutat 30(5):703–714. doi:10.1002/humu.20938 PubMedCrossRefGoogle Scholar
  29. 29.
    Tavtigian SV, Greenblatt MS, Lesueur F et al (2008) IARC Unclassified Genetic Variants Working Group. In silico analysis of missense substitutions using sequence-alignment based methods. Hum Mutat 29(11):1327–1336. doi:10.1002/humu.20892 PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Leonardi E, Murgia A, Tosatto SC (2009) Adding structural information to the von Hippel-Lindau (VHL) tumor suppressor interaction network. FEBS Lett 583(22):3704–3710. doi:10.1016/j.febslet.2009.10.070 PubMedCrossRefGoogle Scholar
  31. 31.
    Forman JR, Worth CL, Bickerton GR et al (2009) Structural bioinformatics mutation analysis reveals genotype-phenotype correlations in von Hippel-Lindau disease and suggests molecular mechanisms of tumorigenesis. Proteins 77(1):84–96. doi:10.1002/prot.22419 PubMedCrossRefGoogle Scholar
  32. 32.
    Gerlinger M, Rowan AJ, Horswell S et al (2012) Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 366(10):883–892. doi:10.1056/NEJMoa1113205 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Mariana Rezende Alves
    • 1
  • Felipe Cavalcanti Carneiro
    • 1
  • André Mourão Lavorato-Rocha
    • 1
  • Walter Henriques da Costa
    • 2
  • Isabela Werneck da Cunha
    • 3
  • Stênio de Cássio Zequi
    • 2
  • Gustavo Cardoso Guimaraes
    • 2
  • Fernando Augusto Soares
    • 3
  • Dirce Maria Carraro
    • 1
  • Rafael Malagoli Rocha
    • 1
    • 3
  1. 1.International Center of Research in Cancer (CIPE)A.C. Camargo Cancer CenterSao PauloBrazil
  2. 2.Urology Division DepartmentA.C. Camargo Cancer CenterSão PauloBrazil
  3. 3.Department of Investigative PathologyA.C. Camargo Cancer CenterSão PauloBrazil

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