Development and verification of new monoclonal orthopedia homeobox (OTP) specific antibodies for pulmonary carcinoid diagnostics

Laura Moonen; Jules L Derks; Lisa M V Lap; Britney J C A Marijnissen; Lisa M Hillen; Michael A den Bakker; Jan H von der Thüsen; Robert-Jan van Suylen; Wim Timens; Maria Bintanel; Eugenia Kuteeva; Anne-Marie C Dingemans; Ernst-Jan M Speel

doi:10.21037/tlcr-22-418

Development and verification of new monoclonal orthopedia homeobox (OTP) specific antibodies for pulmonary carcinoid diagnostics

Transl Lung Cancer Res. 2022 Nov;11(11):2181-2191. doi: 10.21037/tlcr-22-418.

Affiliations

¹ Department of Pathology GROW School for Oncology and Reproduction, Maastricht University Medical Centre, Maastricht, The Netherlands.
² Department of Pulmonary Diseases, GROW School for Oncology and Reproduction, Maastricht University Medical Centre, Maastricht, The Netherlands.
³ Department of Pathology, Maasstad Hospital, Rotterdam, The Netherlands.
⁴ Department of Pathology and Clinical Bioinformatics, Erasmus Medical Center, Rotterdam, The Netherlands.
⁵ Pathology-DNA, Location Jeroen Bosch Hospital, 's-Hertogenbosch, The Netherlands.
⁶ Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
⁷ Atlas Antibodies, Stockholm, Sweden.
⁸ Department of Pulmonary Medicine, Erasmus MC Cancer institute, University Medical Center, Rotterdam, The Netherlands.

Abstract

Background: Orthopedia homeobox (OTP) has shown to be a useful prognostic marker to predict outcome in pulmonary carcinoids, which is also supported by the World Health Organization. However, the discontinuation of the initially used polyclonal antibody and absence of a reliable routinely applicable monoclonal OTP antibody hampers implementation in routine diagnostics. Here, new monoclonal antibodies directed against OTP were developed and verified on formalin-fixed paraffin-embedded tissue of pulmonary neuroendocrine tumors (NETs) for clinical diagnostics.

Methods: OTP specific monoclonal antibodies were produced from mice immunised with a recombinant human OTP protein fragment. Enzyme-linked immunosorbent assay (ELISA) positive hybridomas were evaluated using immunohistochemistry (IHC). Following epitope-mapping and isotyping, purified monoclonal antibodies were validated for IHC in formalin-fixed paraffin-embedded tissues, the optimal dilution was determined, and results were cross validated with the OTP polyclonal antibody (HPA039365, Atlas Antibodies). Staining protocols were optimized on two automated staining platforms and performance was harmonized using a tissue microarray (TMA).

Results: Two clones (CL11222 and CL11225) were selected for purified monoclonal antibody (mAb) production. Intratumor heterogeneity assessment revealed similar performance for both clones. While clone CL11225 displayed a unique epitope compared to those present in the polyclonal antibody, this clone performed most similar to the polyclonal antibody. Cross-platform assessment revealed an excellent agreement for clone CL11225 while clone CL11222 showed somewhat discordant results on Dako.

Conclusions: New monoclonal OTP specific antibodies have been developed and verified on different automated immunohistochemical staining platforms. The OTP specific monoclonal antibodies showed excellent agreement with the often-used polyclonal antibody allowing application in routine diagnostics.

Keywords: Pulmonary carcinoids; monoclonal antibody (mAb); neuroendocrine; orthopedia homeobox (OTP); prognostic.