Virchows Archiv

, Volume 470, Issue 1, pp 99–108 | Cite as

Validation of specificity of antibodies for immunohistochemistry: the case of ROR2

  • Sean S.Q. Ma
  • Claire E. Henry
  • Estelle Llamosas
  • Rupert Higgins
  • Benjamin Daniels
  • Luke B. Hesson
  • Nicholas J. Hawkins
  • Robyn L. Ward
  • Caroline E. Ford
Original Article


The Wnt signalling receptor receptor tyrosine kinase-like orphan receptor 2 (ROR2) is implicated in numerous human cancers. However, there have been conflicting reports regarding ROR2 expression, some studies showing upregulation and others downregulation of ROR2 in the same cancer type. The majority of these studies used immunohistochemistry (IHC) to detect ROR2 protein, without validation of the used antibodies. There appears to be currently no consensus on the antibody best suited for ROR2 detection or how ROR2 expression changes in various cancer types. We examined three commercially available ROR2 antibodies and found that only one bound specifically to ROR2. Another antibody cross-reacted with other proteins, and the third failed to detect ROR2 at all. ROR2 detection by IHC on 107 patient samples using the ROR2 specific antibody showed that the majority of colorectal cancers show loss of ROR2 protein. We found no association between ROR2 staining and poor patient survival, as had been previously reported. These results question the previously reported association between ROR2 and poor patient survival in colorectal cancer. Future studies should use fully validated antibodies when detecting ROR2 protein, as non-specific staining can lead to irrelevant observations and misinterpretations.


ROR2 Immunohistochemistry Colorectal cancer Antibodies 


Compliance with ethical standards

Ethics approval was obtained for all research performed on patient samples. The ethics committee approval numbers are HREC 00113, H00/022 and H04/024, UNSW ethics committee. Informed consent was obtained from all patients participating in the study.


This work was supported by the NSW Cancer Council. Support and funding was also provided by the Translational Cancer Research Network (TCRN) and a UNSW Medicine Faculty Research Grant.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

428_2016_2019_MOESM1_ESM.xlsx (18 kb)
ESM 1 (XLSX 18 kb)
428_2016_2019_Fig6_ESM.gif (65 kb)
Supplementary Figure 1

(GIF 64 kb)

428_2016_2019_MOESM2_ESM.eps (1.6 mb)
High resolution image (EPS 1667 kb)


  1. 1.
    Afzal AR et al. (2000) Recessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2. Nat Genet 25(4):p. 419–p. 422CrossRefGoogle Scholar
  2. 2.
    Begley CG, Buchan AM, Dirnagl U (2015) Robust research: institutions must do their part for reproducibility. Nature 525(7567):p. 25–p. 27CrossRefGoogle Scholar
  3. 3.
    Cancer Genome Atlas, N (2012) Comprehensive molecular characterization of human colon and rectal cancer. Nature 487(7407):p. 330–p. 337CrossRefGoogle Scholar
  4. 4.
    Chen TH et al. (2013) The prognostic significance of APC gene mutation and miR-21 expression in advanced-stage CRC. Color Dis 15(11):p. 1367–p. 1374CrossRefGoogle Scholar
  5. 5.
    Conlin A et al. (2005) The prognostic significance of K-ras, p53, and APC mutations in colorectal carcinoma. Gut 54(9):p. 1283–p. 1286CrossRefPubMedGoogle Scholar
  6. 6.
    Edris B et al. (2012) ROR2 is a novel prognostic biomarker and a potential therapeutic target in leiomyosarcoma and gastrointestinal stromal tumour. J Pathol 227(2):p. 223–p. 233CrossRefGoogle Scholar
  7. 7.
    Egelhofer TA et al. (2011) An assessment of histone-modification antibody quality. Nat Struct Mol Biol 18(1):p. 91–p. 93CrossRefGoogle Scholar
  8. 8.
    Enomoto M et al. (2009) Autonomous regulation of osteosarcoma cell invasiveness by Wnt5a/Ror2 signaling. Oncogene 28(36):p. 3197–p. 3208CrossRefGoogle Scholar
  9. 9.
    Ford CE et al. (2012) The dual role of the novel wnt receptor tyrosine kinase, ROR2, in human carcinogenesis. Int J Cancer 133(4):p. 779–p. 787CrossRefGoogle Scholar
  10. 10.
    Geng M et al. (2012) Loss of Wnt5a and Ror2 protein in hepatocellular carcinoma associated with poor prognosis. World J Gastroenterol 18(12):p. 1328–p. 1338CrossRefPubMedGoogle Scholar
  11. 11.
    He L et al. (2015) The clinical pathological significance of FRAT1 and ROR2 expression in cartilage tumors. Clin Transl Oncol 17(6):p. 438–p. 445CrossRefPubMedGoogle Scholar
  12. 12.
    Henry C et al. (2015a) Expression of the novel wnt receptor ROR2 is increased in breast cancer and may regulate both beta-catenin dependent and independent wnt signalling. J Cancer Res Clin Oncol 141(2):p. 243–p. 254CrossRefPubMedGoogle Scholar
  13. 13.
    Henry C et al. (2015b) Targeting the ROR1 and ROR2 receptors in epithelial ovarian cancer inhibits cell migration and invasion. Oncotarget 6(37):p. 40310–p. 40326Google Scholar
  14. 14.
    Huang J et al. (2015) High ROR2 expression in tumor cells and stroma is correlated with poor prognosis in pancreatic ductal adenocarcinoma. Sci Rep 5:12991CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Jeon CH et al. (2008) Genetic alterations of APC, K-ras, p53, MSI, and MAGE in Korean CRC patients. Int J Color Dis 23(1):p. 29–p. 35CrossRefGoogle Scholar
  16. 16.
    Kobayashi M et al. (2009) Ror2 expression in squamous cell carcinoma and epithelial dysplasia of the oral cavity. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 107(3):p. 398–p. 406CrossRefGoogle Scholar
  17. 17.
    Lara E et al. (2010) Epigenetic repression of ROR2 has a wnt-mediated, pro-tumourigenic role in colon cancer. Mol Cancer:9Google Scholar
  18. 18.
    Lee SE et al. (2013) Prognostic significance of Ror2 and Wnt5a expression in medulloblastoma. Brain Pathol 23(4):p. 445–p. 453CrossRefGoogle Scholar
  19. 19.
    Lipman NS et al. (2005) Monoclonal versus polyclonal antibodies: distinguishing characteristics, applications, and information resources. ILAR J 46(3):p. 258–p. 268CrossRefGoogle Scholar
  20. 20.
    Liu G, Kukuruzinska MA, Xu X (2013) Ror2 may be downregulated in oral squamous cell carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol 116(1):p. 120CrossRefGoogle Scholar
  21. 21.
    Lu BJ et al. (2012) Expression of WNT-5a and ROR2 correlates with disease severity in osteosarcoma. Mol Med Rep 5(4):p. 1033–p. 1036PubMedGoogle Scholar
  22. 22.
    Lugli A et al. (2007) Prognostic significance of the wnt signalling pathway molecules APC, beta-catenin and E-cadherin in CRC: a tissue microarray-based analysis. Histopathology 50(4):p. 453–p. 464CrossRefPubMedGoogle Scholar
  23. 23.
    Ma SSQ et al. (2016) ROR2 is epigenetically inactivated in the early stages of colorectal neoplasia and is associated with proliferation and migration. BMC Cancer 16:508. doi: 10.1186/s12885-016-2576-7 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Mei, H., et al., (2014) High expression of ROR2 in cancer cell correlates with unfavorable prognosis in CRC. Biochem Biophys Res Commun.Google Scholar
  25. 25.
    Michel MC, Wieland T, Tsujimoto G (2009) How reliable are G-protein-coupled receptor antibodies? Naunyn Schmiedeberg's Arch Pharmacol 379(4):p. 385–p. 388CrossRefGoogle Scholar
  26. 26.
    Mikels A, Minami Y, Nusse R (2009) Ror2 receptor requires tyrosine kinase activity to mediate Wnt5A signaling. J Biol Chem 284(44):p. 30167–p. 30176CrossRefGoogle Scholar
  27. 27.
    Nomachi A et al. (2008) Receptor tyrosine kinase Ror2 mediates Wnt5a-induced polarized cell migration by activating c-Jun N-terminal kinase via actin-binding protein filamin a. J Biol Chem 283(41):p. 27973–p. 27981CrossRefGoogle Scholar
  28. 28.
    O'Connell MP et al. (2010) The orphan tyrosine kinase receptor, ROR2, mediates Wnt5A signaling in metastatic melanoma. Oncogene 29(1):p. 34–p. 44CrossRefGoogle Scholar
  29. 29.
    O'Connell MP et al. (2013) Hypoxia induces phenotypic plasticity and therapy resistance in melanoma via the tyrosine kinase receptors ROR1 and ROR2. Cancer Discov 3(12):p. 1378–p. 1393CrossRefPubMedGoogle Scholar
  30. 30.
    Rasmussen NR et al. (2013) Receptor tyrosine kinase-like orphan receptor 2 (Ror2) expression creates a poised state of wnt signaling in renal cancer. J Biol Chem 288(36):p. 26301–p. 26310CrossRefGoogle Scholar
  31. 31.
    Sun B et al. (2015) Up-regulation of ROR2 is associated with unfavorable prognosis and tumor progression in cervical cancer. Int J Clin Exp Pathol 8(1):p. 856–p. 861PubMedGoogle Scholar
  32. 32.
    Taniguchi Y et al. (2010) Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329(5991):p. 533–p. 538CrossRefGoogle Scholar
  33. 33.
    Uhlen M et al. (2015) Proteomics. Tissue-based map of the human proteome. Science 347(6220):p. 1260419CrossRefGoogle Scholar
  34. 34.
    Vogel C, Marcotte EM (2012) Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet 13(4):p. 227–p. 232Google Scholar
  35. 35.
    Wang D (2008) Discrepancy between mRNA and protein abundance: insight from information retrieval process in computers. Comput Biol Chem 32(6):462–468CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Wright TM et al. (2009) Ror2, a developmentally regulated kinase, promotes tumor growth potential in renal cell carcinoma. Oncogene 28(27):p. 2513–p. 2523CrossRefGoogle Scholar
  37. 37.
    Xu XM et al. (2012) DNA alterations of microsatellite DNA, p53, APC and K-ras in Chinese CRC patients. Eur J Clin Investig 42(7):p. 751–p. 759CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Sean S.Q. Ma
    • 1
  • Claire E. Henry
    • 1
  • Estelle Llamosas
    • 1
  • Rupert Higgins
    • 1
  • Benjamin Daniels
    • 2
  • Luke B. Hesson
    • 3
  • Nicholas J. Hawkins
    • 4
  • Robyn L. Ward
    • 4
  • Caroline E. Ford
    • 1
  1. 1.Adult Cancer Program, Level 2, Metastasis Research Group, Lowy Cancer Research Centre and School of Women’s and Children’s HealthUniversity of New South WalesSydneyAustralia
  2. 2.Faculty of Medicine, Medicines Policy Research Unit, Centre for Big Data Research in HealthUNSWSydneyAustralia
  3. 3.Colorectal Cancer Group, Adult Cancer Program, Lowy Cancer Research CentreUNSWSydneyAustralia
  4. 4.University of QueenslandSt. LuciaAustralia

Personalised recommendations