Supplementary Material for: Circulating Tumor DNA Profiling Identifies Actionable Mutations as Prognostic Markers in Advanced Neuroendocrine Tumors

Introduction
Neuroendocrine tumors (NETs) are a rare and heterogeneous group of neoplasms with both clinical and genetic diversity. The clinical applicability of molecular profiling using liquid biopsy for identifying actionable drug targets and prognostic indicators in patients with advanced NETs remains unclear.
Methods
In this study, we utilized a custom-made 37 genes panel of circulating tumor DNA (ctDNA) based on next-generation sequencing (NGS) in 47 patients with advanced NETs.
Results
A total of 223 non-synonymous mutations were identified, with the highest frequencies found in TSC2, JAG2, NOTCH3, KMT2C, and SETD2. NETs originating from the stomach exhibited the highest average mutation frequency, while paragangliomas had the lowest. TERT mutations were associated with significantly higher hazard ratios for both progression-free survival (PFS) and overall survival (OS). Higher blood tumor mutational burden (bTMB) was linked to poor prognosis in Octreotide LAR-treated patients and correlated with higher Ki-67 levels, and alterations in the TM, mTOR, and Notch pathways. Treatment responders had higher bTMB, chromatin remodeling signaling mutation burden, and maximum somatic allele frequency (MSAF). Patients with high MSAF demonstrated better PFS, whereas those with low MSAF had better OS. A strong positive correlation was observed between MSAF levels and mutational burden. Co-occurring mutations in the TM, mTOR, and NOTCH pathways suggested future therapeutic strategies.
Conclusions
We constructed an overview of mutations in 37 genes, linking ctDNA to survival in advanced NETs. TERT mutations, bTMB, and MSAF predict PFS or OS. Co-occurring TM, mTOR, and NOTCH pathway alterations highlight ctDNA's potential in guiding precision medicine.