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

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.