br Discussion Metastatic RCC is a fatal disease with
Discussion Metastatic RCC is a fatal disease with survival often measured in months, rather than years. Surgery alone is the standard of care for patients who present with isolated renal disease at the time of diagnosis. However, approximately 1/3 of these patients will go on to develop clinically detectable metastatic disease, which is then treated with systemic therapies that have been shown to prolong survival by several months . However, if identification of the subset of patients who are at high risk for developing metastatic disease could accurately be determined, systemic therapy could be initiated at the time of diagnosis with the goal of targeting the clinically undetectable micro-metastatic disease. Ultimately, as targeted systemic therapies continue to improve, this could enhance long-term overall survival in this population of patients. To the end of identifying novel molecular targets to diagnose and treat RCC, our microarray studies identified IMP3 (Supplementary Table 1). IMP3 is a cytosolic protein that binds mRNA, and targets specific transcripts for processing within the cell. Although first identified as having high binding affinity for igf2 mRNA, IMP3 also binds to many different mRNAs within the cell, and thereby influences gene expression, including genes involved in cellular migration and proliferation . Interestingly, IMP3 is generally not expressed in normal adult tissues, but has been found to be present in several malignancies to varying degrees including RCC [16,26]. Thus, it has potential as a molecular diagnostic and targeted therapy if its role is determined to be essential for cancer progression. Consistent with this thinking, several prior studies have shown that IMP3 expression is correlated with a poorer prognosis in patients with RCC [14,26]. Therefore, we aimed to assess the relationship between IMP expression, RCC metastatic potential and RCC vascularity as determined by 69 9 enhanced CT. RCC is generally considered to be a vascular tumor both at the primary site and at the sites of metastasis, and its vascularity has been strongly implicated in the tumor’s pathogenesis and invasive potential. Based on our initial investigations using microarray analysis and review of the current literature, we found that the IMP family of proteins may be implicated in RCC tumor virulence, and possibly vascular growth and development within the tumor. Further analysis by real-time RT-PCR (Supplementary Fig. 1) confirmed that IMP3 is highly expressed in RCC, and prompted further study of this protein and its role in the vascularity and metastatic potential of RCC. Several studies have been performed by other groups that indicate the possibility of discriminating renal cell carcinoma subtypes using texture analysis or single phase corticomedullary contrast-enhanced CT [18–21]. These studies also found that CT contrast enhancement was a valid method of representing tumor vascularity in either hetero- or homogeneously contrast-enhanced lesions. In order to directly assess the relationship between IMP3 expression in primary RCC and tumor vascularity, we first had to develop a quantitative outcome measure of tumor vascularity in vivo (Figs. 2 and 4A). Although several approaches to quantify tumor vascularity have been described, a consensus on the best method has not emerged . As we were restricted to protocols that utilize clinical CT scans, we chose a method similar to that which we have described for other clinical conditions [28,29], but acknowledge that this approach still requires formal prospective studies for validation. Nevertheless, we find our contrast CT approach to be safe, feasible and readily translatable if future studies can validate its sensitivity and specificity. One surprising result from our CT study was the lack of correlation between primary RCC tumor size and its vascularity. While the Low contrast enhancing tumors were 2-fold smaller than the Intermediate enhancing tumors as expected, the High contrast enhancing tumors were also significantly smaller than the Intermediate tumors (Fig. 4). Holding to the theory that RCC tumor vascularity is associated with virulence, our interpretation of this finding is that High contrast enhancing primary RCC tumors greater than 200cm3 are incompatible with human life, and thus none were observed in our study. However, consistent with the central hypothesis of this study, we found a strong association between primary RCC vascularity as measured by CT and IMP3 expression (Fig. 4D). Furthermore, IHC assessment of tissue from 11 patients with confirmed metastatic RCC to bone revealed that all of the metastatic tumors had strong IMP3 staining (Fig. 3). In contrast, we did not see strong IHC staining in poorly vascular metastatic bone-prostate cancer biopsies. Although our conclusions are limited by the small sample size, these findings suggest that IMP3 may play a critical role in tumor vascular development and potential to spread.