Supplementary Material for: Reviewing the Osteotropism in Neuroendocrine Tumors: The Role of Epithelial-Mesenchymal Transition

<b><i>Background:</i></b> Neuroendocrine tumors (NETs) metastasize to the bone. However, the incidence, clinical features, management and pathogenesis of bone involvement in NET patients have been poorly investigated. <b><i>Methods:</i></b> We reviewed all published reports of histologically confirmed bone metastatic NETs and explored clinical, radiological, prognostic and therapeutic characteristics in a population of 152 patients. We then evaluated immunohistochemical expression of a panel of eight epithelial-mesenchymal transition (EMT)-related factors including SNAIL, TGF-β1, CTGF, IL-11, PTHrP, EpCAM, CXCR4 and RANK in an independent cohort of 44 archival primary NETs. Biomarker expression was correlated with clinicopathological variables, including skeletal involvement, and tested for survival prediction. <b><i>Results:</i></b> We found that 55% of NET patients with bone metastases were male, with a median age of 55 years at diagnosis. Metastases were restricted to the skeleton in 34% of the NET population, and axial and osteoblastic lesions were prevalent. NETs differently expressed proteins involved in EMT activation. High CXCR4 (p < 0.0001) and low TGF-β1 levels (p = 0.0015) were significantly associated with increased risk of skeletal metastases, suggesting that EMT is implicated in NET osteotropism. By applying an algorithm measuring distinct immunohistochemical predictors of osteotropism on primary tumors, we were able to identify NET patients with bone metastases with a sensitivity and specificity of 91 and 100%, respectively (p < 0.0001). Patients whose primary tumors expressed CTGF (p = 0.0007) as well as the truncated form of EpCAM (p = 0.06) showed shorter survival. <b><i>Conclusion:</i></b> Although underestimated, bone metastases are a prominent feature of NETs, and the tumor expression of EMT markers at diagnosis may predict concurrent or subsequent skeleton colonization.