Evaluation of AFP expression as a predictive marker for response to anti-VEGFR-2 inhibition

Abstract

Introduction: Ramucirumab is a fully human monoclonal IgG1 antibody that specifically targets vascular endothelial growth factor receptor 2 (VEGFR-2). In the phase 3, randomized REACH study in patients with advanced hepatocellular carcinoma (HCC) following first-line sorafenib, Ramucirumab improved median overall survival in a prespecified subgroup of patients who had a baseline a-fetoprotein (AFP) level of 400 ng/ mL or greater. HCC patients with high baseline AFP typically have a very poor prognosis and have tumors that exhibit a more aggressive and invasive phenotype. Relationship between the AFP levels and response to anti-angiogenics (ramucirumab) in HCC is not well understood. Methods: CRISPR, shRNA and siRNA techniques were used to silence AFP in AFP high Huh7 cells to understand its role. To better understand the relationship between baseline AFP levels and response to VEGFR2 inhibition in HCC, we performed in vivo xenograft studies using four human HCC cell lines (HuH-7, PLC/PRF/5, SNU-398 and SK-Hep-1) which had differential mRNA expression of AFP. DC101, a rat surrogate for ramucirumab that specifically targets mouse VEGFR-2, was used for the efficacy studies. Expression of 44 human and mouse angiogenesis-related growth factors were evaluated using Luminex based multi-analyte profiling to better understand the differences in these 4 xenograft models. Results: Protein analysis in both cell lysates and in conditioned media confirmed very high and high AFP levels in HuH7 and PLC/PRF/5 respectively, whereas AFP expression by SNU-398 and SK-Hep-1 was below detectable levels. Silencing of AFP reduced the viability of AFP high Huh-7 cells, suggesting that AFP may play a role in regulating cell proliferation in HCC. DC101 inhibited tumor growth in very high AFP HuH-7 and high-AFP PLC-PRF/5 xenograft models, whereas in the AFP negative SNU-398 and SK-Hep-1 xenograft models, mice treated with DC101 exhibited progressive disease. Histological evaluation indicated that DC101 effectively reduced tumor vessels in both very high AFP (HuH-7) and high AFP (SNU-398) xenografts. To investigate potential efficacy differences between the four xenograft models, we evaluated expression of 44 human and mouse angiogenesis-related growth factors. The results demonstrated differential expression of growth factors including VEGF-C, VEGF-D, FGF-2 and IL-8 depending on the AFP levels, of which some showed similar correlations using bioinformatics analysis of up to 26 cell lines. Future studies will focus on confirming these findings in patient derived xenograft models that have patient history, tumor histological staging and gene data available. Conclusion: These studies offer initial mechanistic insights into the efficacy of ramucirumab in HCC patients with high AFP levels, an area of high unmet clinical need.