Phd in MOLECULAR MEDICINE

Thesis title: Lymph node micrometastases in OSCC: molecular profiling of histologically negative lymph nodes for prognostic and therapeutic purposes

Background: Early-stage oral squamous cell carcinoma (OSCC) (pT1–pT2, pN0) represents a significant portion of oral cavity cancers. Despite primary surgical treatment, up to 30% of early-stage patients experience recurrence, even with histologically negative lymph nodes. This study investigated whether molecular analysis—examining the mutational status of the most frequently mutated genes in oral cancer and the expression of specific miRNAs—could detect occult dissemination and improve risk stratification. Methods: Somatic mutations identified by whole-exome sequencing (WES) in primary tumors from an early-stage OSCC cohort were used as patient-specific markers. Digital PCR was subsequently applied to histologically negative lymph nodes from recurrent (n=13) and non-recurrent (n=6) early stage OSCC patients. Tumor tissue and matched metastatic lymph nodes (N+) from a subgroup of 4 late-stage OSCC patients (pN>0) were used as controls. Additionally, a 4-miRNA expression signature, previously identified as prognostic in head and neck cancer, was assessed in the same tissue cohorts by qPCR and combined with mutational status. Finally, to enhance risk stratification, mutational status and miRNA expression were also longitudinally evaluated in pre- and post-surgery liquid biopsy samples (saliva and blood) from three recurrent patients. Results: By WES analysis, we identified TP53, PIK3CA and CDKN2A mutations in all primary tumor tissues from both recurrent and not recurrent cohorts. Interestingly, in recurrent cohort more than 90% of patients show the same mutations detected in the matched primary tumor, in one or more histologically negative lymph nodes often across multiple cervical levels, with variant allele frequencies (VAFs) of 0.01–1.5%. The same mutations were invariably confirmed in recurrences, independently of the period between the first diagnosis and recurrence development (ranging between 7 months and10 years), underscoring their stability as clonal drivers. By contrast, non-recurrent patients displayed predominantly negative lymph nodes for mutations detected in the matched primary tumors, consistent with long-term disease-free survival (60–132 months); only one showed sporadic, low-level positivity not linked to recurrence. Histologically positive lymph nodes from 4 late stage OSCC patients, have been used a s control confirmed the presence of primary mutation in metastatic nodes, with VAFs proportional to tumour cell content. MiRNAs expression has also been evaluated in the same cohort, demonstrating consistent deregulation of specific transcripts within tumour tissue compared with histologically normal mucosa, even if not always directly correlated with mutational status. Additionally, liquid biopsy samples of 3 early stage OSCC recurrent patients have been also analysed. The results revealed immediate post-surgical clearance, followed by early reappearance of minimal residual disease (MRD) and progressive ctDNA increase preceding clinical recurrence. Mouthwash analysis showed greater sensitivity to locoregional persistence compared with plasma. miRNAs expression in liquid biopsy confirmed the feasibility of detecting circulating small RNAs in serum and saliva, although their dynamics were not always concordant with ctDNA, suggesting that miRNAs may provide complementary biological information rather than strictly overlapping signals. Conclusions: Molecular analysis of histologically negative lymph node of early stage OSCC is a strong predictor of recurrence, digital PCR and liquid biopsy provide complementary tools for detecting occult dissemination, lymph node micrometastases and monitoring MRD, with potential to refine TNM staging and enable personalised management.