Thesis title: High resolution NGS approach for the non-invasive detection of somatic mutations in solid tumors
Head and neck squamous cell carcinomas (HNSCC) are among the most common malignancies worldwide. They are characterised by a high rate of local recurrence and by considerable mortality, often a consequence of late diagnosis. The main risk factors include tobacco, alcohol consumption, and human papillomavirus (HPV) infection. Despite therapeutic advances, particularly in surgery, radiotherapy, and chemotherapy, the prognosis remains poor, and effective strategies for molecular characterisation and monitoring are still needed.
This project aims to develop and clinically validate a targeted Next-Generation Sequencing (NGS) assay for mutational profiling of HPV-negative HNSCC: the H&N chip.
The H&N chip was developed at Eurofins Genoma Group S.r.l. (Rome, Italy), in collaboration with the Istituto Nazionale Tumori Regina Elena (IRE, Rome, Italy), with the aim of providing a sensitive and clinically applicable diagnostic tool. The performance of the H&N chip was evaluated alongside digital PCR (dPCR), and its application was examined in tissue, plasma, and saliva samples.
The H&N chip showed high analytical sensitivity, with a limit of detection of 0.25% established on reference standards. In tissue, it detected variants in the majority of patients, distributed across the three genes included in the chip: TP53, FAT1, and CDKN2A. dPCR confirmed all the evaluable variants and showed high agreement in allele frequency.
In plasma, circulating tumour DNA (ctDNA) was detected in a substantial subset of patients, mostly at low allele frequency. In one case, plasma revealed a sub clonal TP53 variant not detected in the tissue biopsy, consistent with intratumoral heterogeneity. In addition, a preliminary saliva-based analysis supported the feasibility of tumour monitoring in this non-invasive matrix.
Overall, this study demonstrates that the H&N chip is a sensitive and practical assay for detecting variants in HPV-negative HNSCC. Combined with dPCR, it enables mutational profiling across different matrices, with the potential to be integrated into diagnostic and monitoring pathways.