Minimally invasive determination of PDAC subtype and therapy-induced subtype switch by means of circulating cell-free RNA

Abstract

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) comprises two clinically relevant subtypes. Currently, determining the tumor subtype relies on tissue biopsies. Unfortunately, these biopsies are spatially biased, highly invasive, difficult to obtain, and unsuitable for monitoring tumor dynamics. Methods We employed whole transcriptome sequencing (WTS) on circulating cell-free (cf) RNA in plasma samples from patients with well-characterized tumor subtypes. Additionally, quantitative protein mass spectrometry was utilized to identify minimally invasive markers for tumor subtypes. We validated our findings using independent liquid and tissue samples from large clinical trials and investigated treatment-induced subtype dynamics and responses. Results An exploratory analysis of 10 patients (four basal-like and six classical) was conducted using whole transcriptome sequencing (WTS). Following differential transcript abundance analysis and integration with expression data from tumor and non-tumor samples (N > 200), we identified 32 protein-coding subtype-specific cfRNA-defined transcripts. The subtype specificity of these transcripts was validated in two independent tissue cohorts comprising 195 and 250 cases, respectively. Three disease-relevant cfRNA-defined subtype markers (DEGS1, KDELC1, and RPL23AP7) consistently associated with basal-like tumors across all cohorts and were validated using machine learning. Further analysis of these markers using RT-ddPCR in over 160 patient sera and 24 samples from healthy donors revealed their predictive and prognostic value, as well as subtype specificity and therapy-induced dynamics. In both tumor and liquid biopsies, the overexpression of these markers was associated with poor overall and progression-free survival. Moreover, elevated tissue/liquid levels of the identified markers were linked to a poor response to systemic therapy and rapid disease recurrence in resected patients. Conclusion Our data provide support for the clinical significance of cfRNA markers in determining tumor subtypes and monitoring disease recurrence and therapy-induced subtype switches in pancreatic ductal adenocarcinoma (PDAC). Consequently, further validation studies in larger independent cohorts are warranted to confirm the robustness and generalizability of these findings.