Clinical Significance of Heparanase Splice Variant (T5) in Renal Cell Carcinoma: Evaluation by a Novel T5-Specific Monoclonal Antibody

T5 is a novel splice variant of heparanase, an endo-β-D-glucuronidase capable of cleaving heparan sulfate side chains at a limited number of sites. T5 splice variant is endowed with pro-tumorigenic properties, enhancing cell proliferation, anchorage independent growth and tumor xenograft development despite lack of heparan sulfate-degrading activity typical of heparanase. T5 is over expressed in the majority of human renal cell carcinoma biopsies examined, suggesting that this splice variant is clinically relevant. T5 is thought to assume a distinct three-dimensional conformation compared with the wild type heparanase protein. We sought to exploit this presumed feature by generating monoclonal antibodies that will recognize the unique structure of T5 without, or with minimal recognition of heparanase, thus enabling more accurate assessment of the clinical relevance of T5. We provide evidence that such a monoclonal antibody, 9c9, preferentially recognizes T5 compared with heparanase by ELISA, immunoblotting and immunohistochemistry. In order to uncover the clinical significance of T5, a cohort of renal cell carcinoma specimens was subjected to immunostaining applying the 9c9 antibody. Notably, T5 staining intensity was significantly associated with tumor size (p = 0.004) and tumor grade (p = 0.02). Our results suggest that T5 is a functional, pro-tumorigenic entity.T5是乙酰肝素酶（heparanase）的新型剪接变体，而乙酰肝素酶是一类可在有限位点切割硫酸乙酰肝素侧链的内切-β-D-葡糖醛酸糖苷酶。尽管缺乏乙酰肝素酶典型的硫酸乙酰肝素降解活性，T5剪接变体仍具备促肿瘤生成特性，可促进细胞增殖、非锚定依赖性生长以及异种移植瘤的形成。在所检测的多数人类肾细胞癌活检样本中，T5呈过表达状态，提示该剪接变体具有临床相关性。相较于野生型乙酰肝素酶蛋白，T5被推测具有独特的三维构象。本研究旨在利用这一结构特征，制备可特异性识别T5独特结构、仅极少交叉识别乙酰肝素酶的单克隆抗体，以实现对T5临床相关性的精准评估。本研究证实，通过ELISA、免疫印迹（immunoblotting）及免疫组化（immunohistochemistry）实验，单克隆抗体9c9相较于乙酰肝素酶，可优先识别T5。为探究T5的临床意义，研究团队对一组肾细胞癌标本采用9c9抗体进行免疫染色分析。值得注意的是，T5的染色强度与肿瘤大小（p = 0.004）及肿瘤分级（p = 0.02）显著相关。本研究结果表明，T5是一种具备功能活性的促肿瘤生成实体。