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  • Our research in this area

    2019-05-17

    Our research in this area has been focused on understanding the highly vascular nature of RCC, which has been largely attributed to loss of function of the von Hippel-Lindau (VHL) gene and resultant vascular endothelial growth factor (VEGF) over-expression, as it is an early event during tumorgenesis and is the most common cause for inherited RCC [8–10]. However, therapies that specifically target VEGF and its receptor have failed to demonstrate significant efficacy in clinical trials [11,12]. Moreover, our observations in a murine xenograft model of bone metastasis demonstrated that the major difference between a highly vascular RCC cell line (786-O) and a prototypical avascular prostate cancer cell line (PC3) is the presence of large smooth muscle and pericyte lined blood vessels within the tumor [13], suggests that non-VEGF signaling pathways may be more important. To test this hypothesis, we performed a microarray analysis of 786-O vs. PC3 by using Affymetrix GeneChip Human Genome U133 Plus 2.0 Array (Chip Lot# LE23BK05) and whole data set has been documented at NCBI GEO (http://www.ncbi.nlm.nih.gov.eleen.top/geo/query/acc.cgi?token=qngrecewnbothcv&acc=GSE61942) and a summary of the notable findings is presented in Supplementary Table 1. Based on our search criteria for candidate molecular markers of RCC vascularity, we chose to focus on insulin-like growth factor II (IGF-II) mRNA binding protein 3 (IGF2BP3 or IMP3). IMP3 is an oncofetal mRNA-binding protein and has been recently described as an independent prognostic marker for renal cell carcinoma (RCC) distant metastasis, and is associated with shorter survival [14]. It also has been noted in other cancers to be associated with cell motility and trans-endothelial migration [15]. IMP3 is a member of the highly conserved family of proteins that have been found to be associated with mRNA transport, translation and turnover. Functionally, IMPs have been shown to modulate cell proliferation, adhesion, migration and invasion [16]. IMP3 expression is almost exclusively limited to embryonic development, as its expression in most adult tissues is undetectable. However, it has recently been found to have significant expression in malignant adult tissue, including RCC [17]. Although an association between the expression of IMP3 in RCC and prognosis has recently been discovered, the predictive studies of IMP3 in clinical practice have not been evaluated [17]. More specifically, the relationship between IMP3 expression and pre-operative imaging characteristics (i.e. computed tomography (CT), magnetic resonance image (MRI) or digital subtraction angiographic (DSA)) has yet to be investigated. To address this, we evaluated the relationship between IMP3 expression in primary RCC versus tumor vascularity quantified from the pre-operative intravenous ion channel CT scan. We also evaluated the expression of IMP3 via IHC from samples of bone biopsies obtained from patients with metastatic RCC to bone and compared them to patients with metastatic prostate bone disease. The goals of this study were to determine whether vascularity as assesses by contrast CT is correlated to IMP3 expression and if IMP3 expression in tumor samples could be used to stratify metastatic disease risk assessment in RCC patients. These findings could provide evidence to support early-aggressive systemic therapy, which is currently not the standard of care for most patients identified with RCC.
    Materials and methods
    Results We performed immunohistochemistry for IMP3 on RCC and PC retrieval tissues from patients with bone metastases (Fig. 3). All of the 11 RCC bone metastases samples tested were highly positive (+++) for IMP3, while the non-neoplastic cells within the tumor parenchyma did not showed immunostaining for IMP3 (Fig. 3A–D). In comparison, the 3PC bone metastases samples all had low (+) levels of IMP3 immunostaining (Fig. 3E). To directly evaluate the IMP3 as a biomarker of RCC vascularity, we completed a retrospective analysis of 72 patients from ZMU who underwent contrast enhanced CT preoperatively. Table 1 provides a summary of patient characteristics for these 72 patients, including clinical manifestations and pathologic classification. As expected, the most common histologic type of RCC was clear cell (76.4%) and 97.2% were a single lesion. The 33 patients included in the IHC analysis had similar characteristics to the 72 patients as a whole. Fig. 1A and B and 1C–E shows images from a representative patient with low contrast uptake and high contrast uptake on CT respectively and subsequent DSA confirming the presence of large tumor feeding vessels in the high contrast uptake tumors, which were not seen in the low contrast uptake tumors. In order to assess the relationships between RCC primary tumor radiological characteristics and molecular markers, we first quantified the CT data to determine the volumes of the kidney, tumor and vascular vs. non-vascular regions of the tumor (Fig. 4). Interestingly, we found that the tumors with Intermediate enhancement (20–40 HU) were 2-fold larger than Low and High enhancing tumors, and that this increase in tumor size was significant in both vascular and non-vascular regions (Fig. 4B). Moreover, the volume of the Intermediate enhancing RCC accounted for ~70% of the kidney volume, while the Low and High enhancing tumors accounted for only ~40% and ~30% of kidney volume respectively (Fig. 4C). Further group analysis revealed that the High enhancing tumors were ~90% vascular, which was significantly greater than the ~70% vascularity observed in Intermediate tumors, which was significantly greater than the ~30% vascularity of the Low enhancement RCC (Fig. 4C). However, individual tumor volume did not correlate with tumor vascularity (data not shown). Finally, we directly compared the vascularity of the primary RCC tumors determined by CT to their IMP3 expression (Fig. 3D). The results demonstrated a significant correlation between HU and IMP3 immunostaining of the tumors.