Detection of PIK3CA Gene Mutation in Head and Neck Squamous Cell Carcinoma Using Droplet Digital PCR and RT-qPCR

Abstract

Head and neck squamous cell carcinomas (HNSCC) are the seventh cause of human malignancy with low survival rate due to late diagnosis and treatment. Its etiology is diverse; however genetic factors are significant. The most common mutations in HNSCC were found in the genes: <i>PIK3CA</i> (10-12%), <i>BRCA1</i> (6%), and <i>BRCA2</i> (7-9%). In some cases, these biomarkers correlate with recurrences or survival showing a potential of prognostic and predictive value. A total of 113 formalin-fixed paraffin embedded (FFPE) tumor samples were collected from patients with HNSCC (oral cavity: 35 (31.0%); oropharynx: 30 (26.0%); larynx: 48 (43.0%)). We examined <i>PIK3CA</i> H1047R mutation by Real Time PCR (RT-qPCR) and droplet digital PCR (ddPCR). <i>BRCA1</i> and <i>BRCA2</i> mutations were analyzed by RT-qPCR while p16 protein expression was assessed by immunohistochemistry. Finally, we identified HPV infection by RT-qPCR. The relationships between genomic alterations and clinical parameters were assessed using the Yates' corrected Chi-squared test or Fisher's exact test for nominal variables. Kaplan Meier plots were applied for survival analysis. Our results revealed 9 <i>PIK3CA</i> H1047R mutations detected by ddPCR: 8 of them were negative in RT-qPCR. Due to the use of different methods to test the presence of the <i>PIK3CA</i> gene mutation, different treatment decisions might be made. That is why it is so important to use the most sensitive methods available. We confirmed the usefulness of ddPCR in the <i>PIK3CA</i> mutation assessment in FFPE samples.