Integrated Analysis of Cell-Free DNA and Novel Protein Biomarkers for Stratification and Therapy Monitoring in Stage IV Pancreatic Cancer: A Preliminary Study

<b>Background:</b> Given the poor prognosis of metastatic pancreatic adenocarcinoma (mPDAC), closer disease monitoring through liquid biopsy, most frequently based on serial measurements of cell-free mutated <i>KRAS</i> (<i>KRAS</i>^mut cfDNA), has become a highly active research focus, aimed at improving patients' long-term outcomes. However, most of the available data show only a limited predictive and prognostic value of single-parameter-based methods. We hypothesized that a combined longitudinal analysis of <i>KRAS</i>^mut cfDNA and novel protein biomarkers could improve risk stratification and molecular monitoring of patients with mPDAC. <b>Methods:</b> We prospectively collected 160 plasma samples from 47 patients with mPDAC at our institution. Highly sensitive single-target ddPCR assays were employed to detect and quantify <i>KRAS</i>^mut cfDNA. Additionally, analysis of ten protein biomarkers was performed through Enzyme-linked Immunosorbent Assay (ELISA), and Carbohydrate-Antigen 19-9 (CA 19-9) dynamics were registered. <b>Results</b>: <i>KRAS</i>^mut cfDNA was detectable in 37/47 (78.7%) patients throughout the course of study, and CA 19-9 levels were elevated in 40 out of 47 (85.1%) patients. <i>KRAS</i>^mut cfDNA increase at the time of the first follow-up could predict inferior progression-free survival (PFS) (Hazard ratio (HR) = 3.40, <i>p</i> = 0.0003) and overall survival (OS) (HR = 4.91, <i>p</i> < 0.0001). In contrast to CA 19-9 kinetics, which were not predictive of outcome, integrated analysis of <i>KRAS</i>^mut cfDNA combined with six evaluated circulating protein biomarkers allowed basal risk stratification at the time of the first follow-up (HR = 10.2, <i>p</i> = 0.0014). <b>Conclusions</b>: A combined longitudinal analysis of <i>KRAS</i>^mut cfDNA with selected protein biomarkers offers significantly improved prognostic value for patients with mPDAC compared to single-parameter methods. This innovative approach is a step forward in the molecular monitoring of mPDAC and should be validated in further prospective studies.