LYVE-1 upregulation and lymphatic invasion correlate with adverse prognostic factors and lymph node metastasis in neuroblastoma

Neuroblastoma (NB) accounts for 15% of all childhood cancer deaths. The majority of patients have widespread lymphatic and/or haematogenous metastases at diagnosis, but lymphangiogenesis has not been well documented. Sixty-seven NBs were immunostained for the lymphatic endothelial marker, LYVE-1, and the lymphatic density (LD) and lymphatic invasion (LI), were counted in LYVE-1-expressing lymphatics. LYVE-1-stained lymphatic vessels and LI were present in 26/67 (39%) and 14/67 (21%) of the NBs, respectively. Central LD (CLD) and LI were higher in NBs from stage 4 (p = 0.012, p = 0.004, respectively), high-risk group (p = 0.030, p = 0.002), NBs with high mitosis karyorrhexis index (MKI) (p = 0.011, p = 0.005), unfavourable histology group (p = 0.040, p = 0.017) and distant lymph node metastasis (LNM) (p < 0.001 for each). Marginal LD (MLD) was higher in patients with LNM (p < 0.001). CLD and MLD correlated with LI (p < 0.001 each). Total LYVE-1 protein levels, quantified by a sensitive enzyme-linked immunosorbent assay (n = 55), were also higher in NBs from patients with stage 4 disease (p = 0.046), high-risk group (p = 0.028), MYCN-amplified NBs (p = 0.034) and LNM (p = 0.038). Kaplan-Meier analysis showed that the presence of CLD was associated with both worse OS at 5 years (77% [95% CI: 62-87%] versus 60% [95% CI: 32-80%], p = 0.062) and EFS (74% [95% CI: 58-85%] versus 43% [95% CI: 15-69%], p = 0.070) and LI with OS (71% [95% CI: 57-81%] versus 56% [95% CI: 26-78%], p = 0.055). Significant upregulation of LYVE-1 and the presence of LI in patients with stage 4 and high-risk disease, MYCN-amplification and LNM suggests that LYVE-1 may have value as predictors of outcome.