Abstract 4583: Versican accumulation and proteolysis in neuroendocrine tumors
Authors: Babiarz Christopher P. , Emmerich Philip B. , Sprackling Carley M. , Pasch Cheri A. , Clipson Linda , Matkowskyj Kristina A. , Asimakopoulos Fotis , Deming Dustin A. 
Published: August 2019

Introduction: Neuroendocrine tumors (NETs) are a rare, understudied form of human cancer lacking robust preclinical models. Our laboratory has recently demonstrated the importance of the tumor microenvironment (TME) in T-cell exclusion from tumors. Here we examine the accumulation of the immunoregulatory proteoglycan, versican (VCAN), and its immunostimulatory proteolysis product, versikine (Vkine), produced by VCAN cleavage via ADAMTS proteins. Immunotherapeutics are increasingly being studied in NETs and investigation of the immune permissive nature of their TME is warranted.

Methods: Human NET tissue microarrays (TMAs) were obtained from the University of Wisconsin Carbone Cancer Center TSB Biobank, containing human NET tissues across a broad spectrum of disease sites including the lung and gastrointestinal (GI) tract among others. Immunohistochemistry was performed using antibodies to VCAN, Vkine, and CD8+ T-cells. VCAN and Vkine levels were measured using a four-tiered relative intensity system (0/1/2/3), while CD8+ T-cell infiltration was measured by counting the CD8+ T-cells per high power field (hpf). Experimental groups were binned as low if VCAN or Vkine scored 0 or 1 and high if VCAN or Vkine scored 2 or 3.

Results: Across all samples, high VCAN levels were observed in 20% of samples while high Vkine levels were observed in 55% of samples. Within the lung NET subset, which includes small cell lung cancer, 15% of samples were designated VCAN high and 65% were Vkine high. For the GI NET subset, 34% of samples scored as VCAN high while 50% scored as Vkine high. This study identifies a greater amount of Vkine high tumors present in NETs than we previously observed for colorectal, anal, pancreatic, and breast cancers. The proteolytic predominant phenotype (VCAN low and Vkine high) was present in 55% of lung NETs and 31% of GI NETs. CD8+ T cell infiltration correlated with the VCAN proteolysis predominant phenotype (VCAN low/Vkine High - CD8+ T-cell 16/hpf vs. all other VCAN/Vkine phenotypes 6.5/hpf, p&lt;0.001). This trend persisted across all NET specimen types, including lung and GI primary NETs.

Discussion: VCAN accumulation occurs in a subset of NETs, likely leading to immune exclusion and potential resistance to immunotherapies. Excitingly, we demonstrate that a large subset of these cancers has active proteolysis of VCAN in the TME leading to release of the immunostimulatory fragment Vkine. Those NETs with low levels of VCAN and high levels of Vkine are predicted to have a permissive TME for immunotherapies. Further studies are needed to discern the underlying mechanisms resulting in this proteolysis and the signaling consequences with Vkine production. Additionally, this study indicates that VCAN proteolysis should be investigated as a novel biomarker for immunotherapy response for NETs.

Citation Format: Christopher P. Babiarz, Philip B. Emmerich, Carley M. Sprackling, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican accumulation and proteolysis in neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4583.