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Development of a Rapid Analysis Method for Bone Resection Margins for Oral Squamous Cell Carcinoma by Immunoblotting

Abstract

The purpose of this proof-of-principle study was to develop a rapid and approachable method to analyse bone resection margins in patients with oral squamous cell carcinoma (OSCC) in an intraoperative setting, similar to assessing frozen sections of soft tissue. Bone excision and risk of remaining tumour cells could be minimised, thus improving reconstruction measures and facilitating convalescence. Frozen, sawed wafers of porcine bone artificially combined with porcine skin (simulating OSCC properties) were used to develop and evaluate a new molecular method: protein transfer from non-decalcified, sawed wafers onto a membrane stained by immunofluorescence (Tissue-ProtTrans). Tissue-ProtTrans was based on the detection of keratin 5/6 as a marker of tumour cells. The results were compared to standard immunohistochemistry (IHC) and H&E results of the same wafers after decalcification. Tissue-ProtTrans resulted in a total assay time of 3.5 h using the Trans-Blot® Turbo™ Transfer System (Bio-Rad) for protein transfer. Amersham Protran® Premium Nitrocellulose Membranes 0.2 µm (GE Healthcare) were stained with a primary antibody to keratin 5/6 (Dako Agilent) and a secondary antibody labelled with IRDye® 800CW (LI-COR). Visualisation was performed with an infrared laser scanner (Odyssey). Upon comparison, five independent experiments on porcine specimens processed with the Tissue-ProtTrans showed similar results to standard IHC and H&E analysis. In comparison to standard IHC results (requiring several days due to decalcification) Tissue-ProtTrans provided similar results, but was much faster (3.5 h). This highly promising method has good potential for further time reduction and will be suitable for intraoperative assessment.

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Acknowledgements

We want to give special thanks to Sandra Krämer, PhD, Clinic for Thoracic and Cardiovascular Surgery for her help in employing the Trans-Blot® Turbo™ Transfer System (Bio-Rad).

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Correspondence to Till Braunschweig.

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There is no conflict of interest to disclose.

Ethical Approval

We used porcine tissue in this study. All applicable international, national and institutional guidelines for the care and use of animals were followed. We used archival human tissue in this study. (1) Use of archival human pelvic bone was reviewed and approved by the Ethics Committee of the Medical Faculty of the RWTH Aachen University (No. ek-173/06). (2) Use of human oral squamous cell carcinoma tissue obtained from RWTH Aachen centralised Biomaterial Bank (RWTH cBMB). The cBMB was reviewed and approved by the Ethics Committee of the Medical Faculty of the RWTH Aachen University. A mandatory prerequisite for incorporation of a biomaterial sample into RWTH cBMB is the written consent of the donor. Before signing, the donor is informed by a medical doctor about the research project and the intended storage of donated samples and associated data. The important contribution of the donor to biomedical research is addressed (quoted from https://www.cbmb.rwth-aachen.de/en/data-privacy). All procedures performed involving human tissue were in accordance with the ethical standards of the institutional research committee which are comparable with the 1964 Helsinki declaration and its later amendments.

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Haase, C., Lethaus, B., Knüchel-Clarke, R. et al. Development of a Rapid Analysis Method for Bone Resection Margins for Oral Squamous Cell Carcinoma by Immunoblotting. Head and Neck Pathol 12, 210–220 (2018). https://doi.org/10.1007/s12105-017-0856-4

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  • DOI: https://doi.org/10.1007/s12105-017-0856-4

Keywords

  • Oral squamous cell carcinoma
  • Head and neck
  • Immunofluorescence
  • Intraoperative care
  • Frozen section
  • Bone
  • Surgical margin