Cell-free RNA profiling uncovers non-canonical circulating D2 transcript elevation in Bladder Cancer plasma

Background: D2 overexpression has emerged as a recurrent molecular feature across multiple cutaneous malignancies, where it contributes to aberrant Thyroid Hormone (TH) activation and tumor-associated metabolic reprogramming. Liquid biopsy approaches based on circulating cell-free RNA (cfRNA) is emerging as non-invasive strategy to profile gene expression alterations and support dynamic monitoring of transcriptional changes during disease progression. Methods: We analyzed 54 plasma samples from patients with BLadder CAncer (BLCA) alongside an equivalent cohort of healthy control individuals. Circulating D2 transcripts were quantified after RNA isolation using a modified phenol-chloroform extraction protocol adapted for low-input plasma samples to maximize retrieval of circulating RNA. Results: D2 transcripts were readily detectable in plasma and showed significantly higher levels in BLCA patients compared with healthy controls. Circulating expression of classical urothelial markers GATA3 and UPK3A, as well as Epithelial-to-Mesenchymal Transition (EMT)-related genes (E-Cadherin, N-Cadherin, Vimentin), was likewise increased in BLCA plasma. However, correlation analyses revealed that D2 expression varied independently from GATA3 and UPK3A across both tumor and non-tumor groups. Conclusions: These findings demonstrate that D2 is detectably elevated in the circulation of BLCA patients and captures tumor-associated transcriptional alterations that are independent of established urothelial markers. The distinct, non-redundant behavior of circulating D2 supports its potential value as a complementary biomarker for minimally invasive molecular profiling of BLCA. Further studies are required to define its diagnostic performance and clinical applicability.