Expression of p16(Ink4a) protein in pleomorphic adenoma and carcinoma ex pleomorphic adenoma proves diversity of tumour biology and predicts clinical course

Aims The aim of the study is to correlate p16(Ink4a) expression with the clinical courses of pleomorphic adenoma (PA), its malignant transformation (CaexPA) and treatment outcomes. Methods Retrospective analysis (1998-2019) of 47 CaexPA, 148 PA and 22 normal salivary gland samples was performed. PAs were divided into two subsets: clinically 'slow' tumours characterised by stable size or slow growth; and 'fast' tumours with rapid growth rate. Results Positive p16(Ink4a) expression was found in 68 PA and 23 CaexPA, and borderline expression in 80 and 20, respectively. All 22 (100%) normal salivary gland samples presented with no p16(Ink4a) expression. Significant difference in p16(Ink4a) expression was observed between normal tissue, PA and CaexPA (chi(2) (4)=172,19; p=0.0001). The PA clinical subgroups were also evaluated separately, revealing additional statistical relations: 'fast' PA and CaexPA differed significantly in p16Ink4a expression (chi(2) (2)=8.06; p=0.01781) while 'slow' PA and CaexPA did not (chi(2) (2)=3.09; p=0.2129). 3-year, 5-year and 10-year survival among p16(Ink4a) positive CaexPA patients was 100%, 90.56% and 60.37%, respectively, and in CaexPA patients with borderline p16(Ink4a) expression was 90.0%, 73.64% and 22.20%, respectively. Statistically significant difference between expression pattern and survival rate was observed (F Cox test - F (16, 24)=2.31; p=0.03075). Conclusions Our study confirms no p16(Ink4a) expression in normal tissue, but reveals differences in expression between 'fast' and 'slow' PA. We suggest that p16(Ink4a) overexpression is connected to PA proliferation and subsequent malignant transformation to CaexPA. Borderline p16(Ink4a) staining correlates with worse prognosis of CaexPA.