title: Validation of specificity of antibodies for immunohistochemistry: the case of ROR2.
abstract: 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.
authors.affiliation: Adult Cancer Program, Level 2, Metastasis Research Group, Lowy Cancer Research Centre and School of Women's and Children's Health, University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Sean S Q
authors.initials: SS
authors.lastname: Ma
authors.affiliation: Adult Cancer Program, Level 2, Metastasis Research Group, Lowy Cancer Research Centre and School of Women's and Children's Health, University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Henry
authors.affiliation: Adult Cancer Program, Level 2, Metastasis Research Group, Lowy Cancer Research Centre and School of Women's and Children's Health, University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Estelle
authors.initials: E
authors.lastname: Llamosas
authors.affiliation: Adult Cancer Program, Level 2, Metastasis Research Group, Lowy Cancer Research Centre and School of Women's and Children's Health, University of New South Wales, Sydney, NSW, 2052, Australia.
authors.forename: Rupert
authors.initials: R
authors.lastname: Higgins
authors.affiliation: Faculty of Medicine, Medicines Policy Research Unit, Centre for Big Data Research in Health, UNSW, Sydney, Australia.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Daniels
authors.affiliation: Colorectal Cancer Group, Adult Cancer Program, Lowy Cancer Research Centre, UNSW, Sydney, Australia.
authors.forename: Luke B
authors.initials: LB
authors.lastname: Hesson
authors.affiliation: University of Queensland, St. Lucia, Australia.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Hawkins
authors.affiliation: University of Queensland, St. Lucia, Australia.
authors.forename: Robyn L
authors.initials: RL
authors.lastname: Ward
authors.affiliation: Adult Cancer Program, Level 2, Metastasis Research Group, Lowy Cancer Research Centre and School of Women's and Children's Health, University of New South Wales, Sydney, NSW, 2052, Australia. caroline.ford@unsw.edu.au.
authors.forename: Caroline E
authors.initials: CE
authors.lastname: Ford
mesh_terms: D000906:Antibodies / Q000276:immunology; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000378:metabolism* / Q000401:mortality*; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D057050:Receptor Tyrosine Kinase-like Orphan Receptors / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D051153:Wnt Proteins / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics
chemical_list: D000906:Antibodies; C495270:CTNNB1 protein, human; D051153:Wnt Proteins; D051176:beta Catenin; C078717:ROR2 protein, human; D057050:Receptor Tyrosine Kinase-like Orphan Receptors
keywords: Antibodies; Colorectal cancer; Immunohistochemistry; ROR2