FGF4-mediated resistance to combined anti-angiogenic and immunotherapy in esophageal squamous cell carcinoma via suppression of high endothelial venules and tertiary lymphoid structure formation.

4077 Background: Many patients with locally advanced esophageal squamous cell carcinoma (LA-ESCC) fail to achieve a pathological complete response (pCR) following neoadjuvant immunochemotherapy plus anti-angiogenic therapy, which is associated with a poor prognosis. The identification of predictive biomarkers and the elucidation of resistance mechanisms constitute a major challenge. This study aims to identify relevant biomarkers and elucidate the underlying mechanisms to improve therapeutic outcomes. Methods: Exploratory analyses were conducted on 70 untreated LA-ESCC patients from a phase II clinical trial (ChiCTR2100051763) treated with two 21-day cycles of neoadjuvant treatment with intravenous camrelizumab (200mg), albumin-paclitaxel (260mg/m 2 ), carboplatin (AUC=5) on day 1, and oral apatinib (250mg) on days 1-14, followed by the third cycle without apatinib. Pre-treatment tumor tissues were subjected to transcriptomic and whole-exome sequencing to identify differential pathways and genetic alterations between patients achieving pCR and non-pCR. Candidate biomarkers were further validated by immunohistochemistry. In vitro and in vivo experiments were employed to elucidate the mechanisms underlying treatment resistance. Results: Pathway analysis identified significant enrichment of the fibroblast growth factor receptor (FGFR) signaling pathway in non-pCR patients, with FGF4 as the most upregulated factor. FGF4 alterations were primarily gene amplifications, occurring in 40% of cases. FGF4 expression predicted treatment resistance (non-pCR) with an AUC of 0.857 (95% CI: 0.744–0.97). Multiplex immunohistochemistry revealed that tumors overexpressing FGF4 exhibited impaired vascular normalization, evidenced by significantly reduced pericyte coverage and basement membrane integrity. These tumors also showed lower densities of high endothelial venules (HEVs), CD8+ T cells, and tertiary lymphoid structures (TLSs) following combination therapy. In vivo experiments confirmed that FGF4 overexpression led to decreased pericyte and basement membrane coverage, reduced HEV density, and diminished CD8+ T cell infiltration. Notably, pharmacological inhibition of FGFR signaling reduced tumor growth and increased the pCR rate, effectively reversing the resistance mediated by FGF4. Conclusions: Aberrant FGF4 expression drives resistance to combined immunochemotherapy and anti-angiogenic therapy by impeding vascular normalization, which subsequently suppresses the formation of HEVs and tertiary TLSs. Targeting FGFRs might be a promising strategy to overcome FGF4-driven treatment resistance in ESCC. Clinical trial information: ChiCTR2100051763.