Renal Cell Carcinoma Bone Metastasis Research Articles

Dysregulated HAI-2 Plays an Important Role in Renal Cell Carcinoma Bone Metastasis through Ligand-Dependent MET Phosphorylation.

MET, a c-met proto-oncogene product and hepatocyte growth factor (HGF) receptor, is known to play an important role in cancer progression, including bone metastasis. In a previous study, we reported increased expression of MET and matriptase, a novel activator of HGF, in bone metastasis. In this study, we employed a mouse model of renal cell carcinoma (RCC) bone metastasis to clarify the significance of the HGF/MET signaling axis and the regulator of HGF activator inhibitor type-2 (HAI-2). Luciferase-transfected 786-O cells were injected into the left cardiac ventricle of mice to prepare the mouse model of bone metastasis. The formation of bone metastasis was confirmed by whole-body bioluminescent imaging, and specimens were extracted. Expression of HGF/MET-related molecules was analyzed. Based on the results, we produced HAI-2 stable knockdown 786-O cells, and analyzed invasiveness and motility. Expression of HGF and matriptase was increased in bone metastasis compared with the control, while that of HAI-2 was decreased. Furthermore, we confirmed increased phosphorylation of MET in bone metastasis. The expression of matriptase was upregulated, and both invasiveness and motility were increased significantly by knockdown of HAI-2. The significance of ligand-dependent MET activation in RCC bone metastasis is considered, and HAI-2 may be an important regulator in this system.

Multidisciplinary Approach in the Treatment of Malignant Bone Tumors

Abstract Introduction. There are two types of tumor lesions at bone level: primary tumors and bone metastasis. These lesions cause a decrease in the structural strength of the bone that may lead to fracture. The goal of the orthopedic treatment is to resect the tumor, alleviate symptomatology, and restore the function of the limb. The orthopedic treatment consists of two phases: tumor resection and limb reconstruction. Treatment of such lesions implies extensive preoperative investigations to determine the local and general extent of the tumor and procedures aimed to facilitate resection. Case presentation. We present the case of a 58-year-old male, diagnosed with clear cell renal carcinoma three years before and treated by nephrectomy. Two months after surgery, the patient started reporting moderate pain in the hip, which gradually intensified. He was first seen in Foişor Hospital in December 2018 when a pathologic bone fracture in the trochanteric region of the right femur was discovered at the radiologic exam. This was supposed to be a bone metastasis from the previously operated renal cell carcinoma. The MRI investigation showed the presence of a large, polylobed bone tumor with an inhomogeneous structure, sized 80/ 72/ 82 mm extending in the adjacent soft tissue. Bone scintigraphy of entire skeleton revealed no other lesions and abdominal MRI exam revealed neither the recurrence of the tumor or the presence of abdominal metastasis. Significant vascularization of the tumor, from branches of the deep femoral artery, was evidenced on the Angio Computer Tomography. Due to the known profuse bleeding of such lesions, an angiography with embolization was also performed. Embolization was done with PVA particles, with a lack of post-embolization load. The Angio Computer Tomography scan performed at three weeks after embolization showed a reduction in size of the tumor. In March 2018 patient underwent bone resection and reconstruction with a G.M.R.S. tumor prosthesis. Resection was performed in a complex team involving also two vascular surgeons. The pathology exam confirmed it was a renal cell carcinoma bone metastasis. Results. At one week after the surgical procedure, the operative wound was healing, the patient had no pain, and he was gradually regaining ambulation. Conclusion. Surgical treatment of such neoplastic pathology is difficult and involves a multidisciplinary approach consisting of disciplines such as oncology, orthopedics, vascular surgery, and pathology. It also implies a significant contribution from radiology and interventional radiology.

Identification of a novel biomarker signature associated with risk for bone metastasis in patients with renal cell carcinoma.

When renal cell carcinoma (RCC) metastasizes to bone (a frequent site of systemic spread of this cancer) it becomes highly resistant to radiation therapy and chemotherapy. A better understanding of the biology of bone metastasis in RCC may permit to identify biomarkers for early detection of subclinical disease and better stratification of patients prior to treatment. We therefore investigated in this study, using a multiplex real-time RT-PCR assay, the expression of a panel of 16 biomarkers involved in angiogenesis and tumor invasion; the panel was applied to primary tumors and normal tissues obtained from clear-cell RCC patients with and without bone metastases. We identified a novel combination of biomarkers associated with the risk of bone metastasis. Among the transcripts of the genes studied, VEGFR-1, VEGFR-2, HIF-1alpha, uPA , and PA I-1 overexpression in tumor tissues was significantly associated with the presence of bone metastasis (p=0.02, p=0.02, p<0.0001, p=0.04, and p=0.03, respectively). No differences were found in the expression of these transcripts in the corresponding normal tissues. This preliminary study provides a promising tool that may help in the management of RCC patients with bone metastasis. Indeed, these predictive markers could be useful to identify subclinical disease, improve staging, and guide treatment decisions.

Calcium-sensing receptor (CaSR) promotes development of bone metastasis in renal cell carcinoma.

Bone metastasis is an important prognostic factor in renal cell carcinoma (RCC). The calcium-sensing receptor (CaSR) has been associated with bone metastasis in several different malignancies. We analyzed the impact of CaSR in bone metastasis in RCC in vitro and in vivo. The RCC cell line 786-O was stably transfected with the CaSR gene and treated with calcium alone or in combination with the CaSR antagonist NPS2143. Afterwards migration, adhesion, proliferation and prominent signaling molecules were analyzed. Calcium treated CaSR-transfected 768-O cells showed an increased adhesion to endothelial cells and the extracellular matrix components fibronectin and collagen I, but not to collagen IV. The chemotactic cell migration and proliferation was also induced by calcium. The activity of SHC, AKT, ERK, P90RSK and JNK were enhanced after calcium treatment of CaSR-transfected cells. These effects were abolished by NPS2143. Development of bone metastasis was evaluated in vivo in a mouse model. Intracardiac injection of CaSR-transfected 768-O cells showed an increased rate of bone metastasis. The results indicate CaSR as an important component in the mechanism of bone metastasis in RCC. Therefore, targeting CaSR might be beneficial in patients with bone metastatic RCC with a high CaSR expression.

BIGH3 Promotes Osteolytic Lesions in Renal Cell Carcinoma Bone Metastasis by Inhibiting Osteoblast Differentiation

BACKGROUND: Bone metastasis is common in renal cell carcinoma (RCC), and the lesions are mainly osteolytic. The mechanism of bone destruction in RCC bone metastasis is unknown. METHODS: We used a direct intrafemur injection of mice with bone-derived 786-O RCC cells (Bo-786) as an in vivo model to study if inhibition of osteoblast differentiation is involved in osteolytic bone lesions in RCC bone metastasis. RESULTS: We showed that bone-derived Bo-786 cells induced osteolytic bone lesions in the femur of mice. We examined the effect of conditioned medium of Bo-786 cells (Bo-786 CM) on both primary mouse osteoblasts and MC3T3-E1 preosteoblasts and found that Bo-786 CM inhibited osteoblast differentiation. Secretome analysis of Bo-786 CM revealed that BIGH3 (Beta ig h3 protein), also known as TGFBI (transforming growth factor beta-induced protein), is highly expressed. We generated recombinant BIGH3 and found that BIGH3 inhibited osteoblast differentiation in vitro. In addition, CM from Bo-786 BIGH3 knockdown cells (786-BIGH3 KD) reduced the inhibition of osteoblast differentiation compared to CM from vector control. Intrafemural injection of mice with 786-BIGH3 KD cells showed a reduction in osteolytic bone lesions compared to vector control. Immunohistochemical staining of 18 bone metastasis specimens from human RCC showed strong BIGH3 expression in 11/18 (61%) and moderate BIGH3 expression in 7/18 (39%) of the specimens. CONCLUSIONS: These results suggest that suppression of osteoblast differentiation by BIGH3 is one of the mechanisms that enhance osteolytic lesions in RCC bone metastasis, and raise the possibilty that treatments that increase bone formation may improve therapy outcomes.

Risk factors for bone metastasis from renal cell cancer

ObjectiveThe prognosis for renal cell carcinoma (RCC) is related to a high rate of metastasis, including 30% of bone metastasis. In this study, we investigate the correlation between diverse clinical factors and bone metastases secondary from renal cell cancer (RCC), and to identify potential risk factors for bone metastasis in newly diagnosed patients and those who have already received treatment. MethodsThe clinical data of 372 patients with RCC were reviewed from January 2000 to August 2016. The correlations between age, gender, histopathologic types, alkaline phosphotase (ALP), CEA, AFP, CA-125, CA-153, CA-199, calcium, hemoglobin (HB) and bone metastases were analyzed. And the risk factors for bone metastases in RCC were identified by multivariate logistic regression analysis. The cutoff value, sensitivity and specificity of the independent correlation factors were calculated by receiver operating characteristic (ROC) curve. ResultsThe bone is the second to the lung as a distant metastasis target site in patients with RCC. Thirty eight individuals were identified with bone metastases. Of these patients, significantly higher levels of ALP, calcium, HB were found than those without bone metastasis (P < 0.05, respectively). No significant differences were detected in CEA, AFP, CA-125, CA-153, CA-199, age, gender and histopathologic types between patients with and without bone metastases (P > 0.05, respectively). Multivariate logistic regression analysis indicated that ALP, calcium and HB were independent risk factors correlated with bone metastasis (P < 0.05, respectively). ROC curves demonstrated these factors had comparable accuracy at predicting bone metastasis (AUC were 0.749, 0.633 and 0.665, respectively). The cutoff values of ALP, calcium and HB were 105.5 U/L, 2.615mmol/L and 111.5g/L, respectively. The sensitivities of them were 57.9%, 36.8% and 71.1% for predicting bone metastasis, with specificities of 83.5%, 95.2% and 65.3%, respectively. ConclusionBased on our study, the concentrations of ALP, calcium and HB were potentially risk factors for bone metastasis in patients with RCC. For newly diagnosed patients, if the values of ALP>105.5U/L, calcium>2.615mmol/L and HB<111.5g/L were detected, intensive monitoring and bone scanning are warranted for them.

Comparative efficacy and safety of bone-modifying agents for the treatment of bone metastases in patients with advanced renal cell carcinoma: a systematic review and meta-analysis.

ObjectiveTo determine the comparative efficacy and safety of all available bone-modifying agents (BMAs) for the treatment of bone metastases in patients with advanced renal cell carcinoma (RCC).ResultsThree studies (259 patients) were identified for the systematic review. Two studies that compared zoledronic acid with placebo or no zoledronic acid showed that zoledronic acid reduced the risk of skeletal-related events (SREs) by 68% (pooled hazard ratio [HR]: 0.32; 95% confidence interval [CI]: 0.19–0.55). The quality of evidence was moderate. The incidence of serious adverse events (AEs) was identical (80%) for both treatment arms in one study and not reported in the other study. In the third study that compared denosumab and zoledronic acid among patients with solid tumors or myeloma, a post-hoc subgroup analysis with individual patient data (155 patients) showed an HR of 0.71 (95% CI: 0.43–1.17) for SREs and a risk ratio of 0.86 (95% CI: 0.68–1.08) for serious AEs for denosumab compared to zoledronic acid.Materials and MethodsWe searched the MEDLINE database, Cochrane Library, WHO International Clinical Trials Registry Platform, and ClinicalTrials.gov database up to January 2017 without language restriction. Only randomized controlled trials were included. When relevant data were missing, we contacted the investigators of the original study. The Grading of Recommendation Assessment, Development, and Evaluation approach was used to assess the evidence certainty.ConclusionsOur moderate-quality evidence indicates that zoledronic acid significantly reduces SREs risk among patients with bone metastases of RCC.

Comparative efficacy and safety of bone-modifying agents for the treatment of bone metastases in patients with advanced renal cell carcinoma: A systematic review and meta-analysis.

e16067 Background: Bone-modifying agents (BMA) have been well-demonstrated to be effective for preventing and inhibiting skeletal-related events (SRE) in patients with bone metastases of breast or prostate cancer. However, the role of BMA treatment has not yet been clearly defined in patients with bone metastases of renal cell carcinoma (RCC). We, therefore, conducted a systematic review and meta-analysis to evaluate the efficacy and safety of BMA in patients with bone metastases of RCC. Methods: Literature search was conducted on MEDLINE, the Cochrane Central Register of Controlled Trials, the WHO International Clinical Trials Registry Platform, and ClinicalTrials.gov to identify randomized controlled trials of BMA for the treatment of bone metastases in RCC patients. The primary outcomes were SRE and serious adverse events (AEs). Hazard ratios (HRs) were calculated with a random effects model. The Grading of Recommendation Assessment, Development, and Evaluation (GRADE) approach was used to assess the certainty of the evidence. This review was prospectively registered on PROSPERO (No. CRD42016032742). Results: Three studies (259 patients) were identified for the systematic review. Two studies that compared zoledronic acid with placebo or non-zoledronic acid showed that zoledronic acid reduced the SRE risk by 68% (HR 0.32; 95% confidence interval (CI) 0.19–0.55; P &lt; 0.0001). The quality of evidence was moderate. No serious osteonecrosis was reported in both studies. The incidence of serious AEs was identical (80%) on both treatment arms in one study and not reported in the other study. In the remaining study, which compared denosumab with zoledronic acid, analyses of the individual patient data shared through Amgen showed a favorable trend for denosumab in terms of SRE (HR 0.71; 95% CI 0.43–1.17) and serious AEs (risk ratio 0.86; 95% CI 0.68–1.08), but this trend did not reach statistical significance. Conclusions: The moderate-quality evidence indicates that zoledronic acid significantly reduces the risk of SRE among patients with bone metastases of RCC.

Autocrine and paracrine STIP1 signaling promote osteolytic bone metastasis in renal cell carcinoma.

Bone metastases are responsible for some of the most devastating complications of renal cell carcinoma (RCC). However, pro-metastatic factors leading to the highly osteolytic characteristics of RCC bone metastasis have barely been explored. We previously developed novel bone-seeking RCC cell lines by the in vivo selection strategy and performed a comparative proteome analysis on their total cell lysate. Here, we focused on STIP1 (stress-induced phosphoprotein 1), the high up-regulated protein in the bone-seeking cells, and explored its clinical relevance and functions in RCC bone metastasis. We observed high levels of both intracellular and extracellular STIP1 protein in bone metastatic tissue samples. Elevated STIP1 mRNA in the primary RCC tumors remarkably correlated with worse clinical outcomes. Furthermore, both human recombinant STIP1 protein and anti-STIP1 neutralizing antibody were used in the functional studies. We found that 1) STIP1 protein on the extracellular surface of tumor cells promoted the proliferation and migration/invasion of RCC tumor cells through the autocrine STIP1-ALK2-SMAD1/5 pathway; and 2) STIP1 protein secreted into the extracellular tumor stromal area, promoted the differentiation of osteoclasts through the paracrine STIP1-PrPc-ERK1/2 pathway. Increased cathepsin K (CTSK), the key enzyme secreted by osteoclasts to degrade collagen and other matrix proteins during bone resorption was further detected in the differentiated osteoclasts. These results provide evidence of the great potential of STIP1 as a novel biomarker and therapeutic target in RCC bone metastasis.

Bone Metastasis from Renal Cell Carcinoma

About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastasis that are often osteolytic and cause substantial morbidity, such as pain, pathologic fracture, spinal cord compression and hypercalcemia. The presence of bone metastasis in RCC is also associated with poor prognosis. Bone-targeted treatment using bisphosphonate and denosumab can reduce skeletal complications in RCC, but does not cure the disease or improve survival. Elucidating the molecular mechanisms of tumor-induced changes in the bone microenvironment is needed to develop effective treatment. The “vicious cycle” hypothesis has been used to describe how tumor cells interact with the bone microenvironment to drive bone destruction and tumor growth. Tumor cells secrete factors like parathyroid hormone-related peptide, transforming growth factor-β and vascular endothelial growth factor, which stimulate osteoblasts and increase the production of the receptor activator of nuclear factor κB ligand (RANKL). In turn, the overexpression of RANKL leads to increased osteoclast formation, activation and survival, thereby enhancing bone resorption. This review presents a general survey on bone metastasis in RCC by natural history, interaction among the immune system, bone and tumor, molecular mechanisms, bone turnover markers, therapies and healthcare burden.

Combination with third-generation bisphosphonate (YM529) and interferon-alpha can inhibit the progression of established bone renal cell carcinoma.

The aim of this study was to investigate whether the third-generation nitrogen-containing bisphosphonate (YM529) can inhibit the progression of established bone renal cell carcinoma (RCC) and to elucidate its mechanism. Antiproliferative effect and apoptosis induction of RCC cells and mouse osteoclasts by YM529 and/or interferon-alpha (IFN-α) were evaluated in vitro using cell counting and in vivo using soft X-ray, the TUNEL method and tartrate-resistant acid phosphatase stain. For the in vivo study, male athymic BALB/cA Jc1-nu nude mice bearing human RCC cell line RBM1-IT4 cells were treated with YM529 and/or IFN-α. The biological activity of osteoclasts was evaluated using the pit formation assay. The antiangiogenetic effect by YM529 and/or IFN-α was analyzed using micro-vessel density and in situ mRNA hybridization. Osteoclast number in bone tumors was decreased in YM529-treated mouse. YM529 also inhibited osteoclast activity and proliferation in vitro, whereas basic fibroblast growth factor expressions and micro-vessel density within tumors were inhibited by IFN-α. Neither YM529 nor IFN-α alone significantly inhibited the growth of established bone metastatic tumors. Combined treatment with YM529 and IFN-α may be beneficial in patients with human RCC bone metastasis. Their effects are mediated by osteoclast recruitment inhibition and inactivation by YM529 and antiangiogenesis by IFN-α.

Three-dimensional (3D) culture of bone-derived human 786-O renal cell carcinoma retains relevant clinical characteristics of bone metastases

Bone metastases from renal cell carcinoma (RCC) are typically lytic, destructive, and resistant to treatment regimens. Current in vitro models for studying metastasis introduce artifacts that limit their usefulness. Many features of tumors growing in bone are lost when human RCC cells are cultured in two-dimensional (2D) plastic substrata. In this study, we established that RCC spheroids, consisting of aggregates of cells, can be grown in a three-dimensional (3D) hyaluronate hydrogel-based culture system. The bone-derived human 786-O RCC subline proliferated and survived long term in these hydrogels. Additionally, RCC spheroids in 3D hydrogels demonstrated lower proliferation rates than their counterparts grown in 2D. Overall, gene expression patterns of RCC spheroids in 3D more closely mimicked those observed in vivo than did those of cells grown in 2D. Of particular importance, selected adhesion molecules, angiogenesis factors, and osteolytic factors that have been shown to be involved in RCC bone metastasis were found to be expressed at higher levels in 3D than in 2D cultures. We propose that the 3D culture system provides an improved platform for RCC bone metastasis studies compared with 2D systems.

Development of a realistic in vivo bone metastasis model of human renal cell carcinoma

About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastases. The incidence of RCC is increasing and bone metastatic RCC merits greater focus. Realistic preclinical bone metastasis models of RCC are lacking, hampering the development of effective therapies. We developed a realistic in vivo bone metastasis model of human RCC by implanting precision-cut tissue slices under the renal capsule of immunodeficient mice. The presence of disseminated cells in bone marrow of tissue slice graft (TSG)-bearing mice was screened by human-specific polymerase chain reaction and confirmed by immunohistology using human-specific antibody. Disseminated tumor cells in bone marrow of TSG-bearing mice derived from three of seven RCC patients were detected as early as 1 month after tissue implantation at a high frequency with close resemblance to parent tumors (e.g., CAIX expression and high vascularity). The metastatic patterns of TSGs correlated with disease progression in patients. In addition, TSGs retained capacity to metastasize to bone at high frequency after serial passaging and cryopreservation. Moreover, bone metastases in mice responded to Temsirolimus treatment. Intratibial injections of single cells generated from TSGs showed 100 % engraftment and produced X-ray-visible tumors as early as 3 weeks after cancer cell inoculation. Micro-computed tomography (μCT) and histological analysis revealed osteolytic characteristics of these lesions. Our results demonstrated that orthotopic RCC TSGs have potential to develop bone metastases that respond to standard therapy. This first reported primary RCC bone metastasis model provides a realistic setting to test therapeutics to prevent or treat bone metastases in RCC.

High calcium concentration in bones promotes bone metastasis in renal cell carcinomas expressing calcium-sensing receptor

BackgroundThe prognosis for renal cell carcinoma (RCC) is related to a high rate of metastasis, including 30% of bone metastasis. Characteristic for bone tissue is a high concentration of calcium ions. In this study, we show a promoting effect of an enhanced extracellular calcium concentration on mechanisms of bone metastasis via the calcium-sensing receptor (CaSR) and its downstream signaling molecules.MethodsOur analyses were performed using 33 (11/category) matched specimens of normal and tumor tissue and 9 (3/category) primary cells derived from RCC patients of the 3 categories: non-metastasized, metastasized into the lung and metastasized into bones during a five-year period after nephrectomy. Expression of CaSR was determined by RT-PCR, Western blot analyses and flow cytometry, respectively. Cells were treated by calcium and the CaSR inhibitor NPS 2143. Cell migration was measured in a Boyden chamber with calcium (10 μM) as chemotaxin and proliferation by BrdU incorporation. The activity of intracellular signaling mediators was quantified by a phospho-kinase array and Western blot.ResultsThe expression of CaSR was highest in specimens and cells of patients with bone metastases. Calcium treatment induced an increased migration (19-fold) and proliferation (2.3-fold) exclusively in RCC cells from patients with bone metastases. The CaSR inhibitor NPS 2143 elucidated the role of CaSR on the calcium-dependent effects. After treatment with calcium, the activity of AKT, PLCγ-1, p38α and JNK was clearly enhanced and PTEN expression was almost completely abolished in bone metastasizing RCC cells.ConclusionsOur results indicate a promoting effect of extracellular calcium on cell migration and proliferation of bone metastasizing RCC cells via highly expressed CaSR and its downstream signaling pathways. Consequently, CaSR may be regarded as a new prognostic marker predicting RCC bone metastasis.

Cadherin-11 in Renal Cell Carcinoma Bone Metastasis

Bone is one of the common sites of metastases from renal cell carcinoma (RCC), however the mechanism by which RCC preferentially metastasize to bone is poorly understood. Homing/retention of RCC cells to bone and subsequent proliferation are necessary steps for RCC cells to colonize bone. To explore possible mechanisms by which these processes occur, we used an in vivo metastasis model in which 786-O RCC cells were injected into SCID mice intracardially, and organotropic cell lines from bone, liver, and lymph node were selected. The expression of molecules affecting cell adhesion, angiogenesis, and osteolysis were then examined in these selected cells. Cadherin-11, a mesenchymal cadherin mainly expressed in osteoblasts, was significantly increased on the cell surface in bone metastasis-derived 786-O cells (Bo-786-O) compared to parental, liver, or lymph node-derived cells. In contrast, the homing receptor CXCR4 was equivalently expressed in cells derived from all organs. No significant difference was observed in the expression of angiogenic factors, including HIF-1α, VEGF, angiopoeitin-1, Tie2, c-MET, and osteolytic factors, including PTHrP, IL-6 and RANKL. While the parental and Bo-786-O cells have similar proliferation rates, Bo-786-O cells showed an increase in migration compared to the parental 786-O cells. Knockdown of Cadherin-11 using shRNA reduced the rate of migration in Bo-786-O cells, suggesting that Cadherin-11 contributes to the increased migration observed in bone-derived cells. Immunohistochemical analysis of cadherin-11 expression in a human renal carcinoma tissue array showed that the number of human specimens with positive cadherin-11 activity was significantly higher in tumors that metastasized to bone than that in primary tumors. Together, these results suggest that Cadherin-11 may play a role in RCC bone metastasis.

Management of Painful Pelvic Bone Metastasis of Renal Cell Carcinoma Using Embolization, Radio-frequency Ablation, and Cementoplasty: A Prospective Evaluation of Efficacy and Safety

To measure the impact on pain relief and patient quality of life using embolization radio-frequency ablation and cementoplasty (ERC) for local combination therapeutic management of painful pelvic bone metastasis of renal cell carcinoma (RCC). This prospective monocentric registry was approved by our Local Institutional Review Board. Between January 2008 and January 2013, all consecutive patients who fully met the inclusion criteria were enrolled in the ERC-procedure prospective registry. They were assigned to follow-up at discharge and again at 1 and 6 months. Efficacy was evaluated using a pain visual analog scale (VAS), and narcotic consumption and quality of life were assessed using the Brief Pain Inventory questionnaire. Fifty-two patients were enrolled, among whom 58 lesions were treated. Technical success was obtained in all procedures. The median VAS score decreased from 7 ± 1.4 (ranges 5-10) at baseline to 3 ± 1.5 (ranges 0-6) at discharge, 2 ± 1.5 (ranges 0-5) at 1 month (p < 0.0001), and 2 ± 1.6 (ranges 0-5) at 6 months. In 28 patients (54 %), narcotic consumption was halved at discharge and halved in 40 (77 %) patients at 1 and 6 months compared with baseline. Five patients had complete pain relief at 1 month. A major improvement in quality of life, especially regarding mood and motion, was observed in all patients. This specific approach to painful bone metastasis is efficient and safe and yields sustained results. The ERC procedure could be suggested for patients with RCC bone metastasis.

Bone-Targeted Therapies for Elderly Patients with Renal Cell Carcinoma: Current and Future Directions

Bone metastases are very common in advanced renal cell carcinoma (RCC) and can have a huge impact on quality of life by leading to skeletal-related events (SREs), including pain, pathologic fractures and spinal cord compression with need for surgery or radiotherapy. Because of their osteolytic aspect and biologic behaviour, these SREs are more common in patients with bone metastases from RCC than from other malignancies. As overall survival is increased by new anti-angiogenic drugs like tyrosine kinase inhibitors and mammalian target of rapamycin inhibitors, the incidence of SREs is rising, making the clinical management of bone metastases in RCC ever more important, especially in the more vulnerable elderly patient. In this review we discuss the current advances and future directions in bone-targeted therapies in patients with RCC with a special focus on the elderly population. Recently, two bone-targeted agents have been approved in the prevention of SREs in advanced RCC: zoledronic acid and denosumab. To date, there is no specific data on the use of bisphosphonates or denosumab in the elderly and specific studies in this setting are warranted. We compare the available evidence for the use and implications of both agents in the elderly patient and give general information on safety concerns that could be more important in these patients.

Abstract B9: Mouse model of primary human renal cell carcinoma metastasis to bone

Abstract More than a third of patients with renal cell carcinoma (RCC) will die from disease and thus renal cell carcinoma has the highest mortality rate of the genitourinary cancers. Localized RCC can be cured by surgery but life expectancy is short for the metastatic disease. Thirty % of RCC metastasizes to bone and these metastases are especially debilitating and resistant to therapy. As the incidence of RCC is increasing, bone metastatic renal cancer merits greater focus. However, the development of effective therapies for RCC bone metastasis is hampered by lack of realistic pre-clinical models. Recent studies have shown that tumorgrafts are the most realistic models of solid tumors that are highly predictive of therapeutic response. We have improved this methodology by implanting precision-cut tumor tissue slices rather than minced tissue chunks into immunodeficient mice. Our aim is to develop a bone metastasis model using tissue slice tumorgrafts (TSGs) derived from fresh RCC specimens rather than established RCC cell lines that do not mimic natural history of metastases. Methods: Cores (8-mm diameter) of fresh renal cancer were obtained from nephrectomy specimens and precision-cut at 300-μm using a tissue slicer. Intact tissue slices were then implanted under the renal capsules of immunodeficient mice. The presence of disseminated cells in the bone marrow of graft-bearing mice was screened by qPCR (polymerase chain reaction) for human-specific markers. Primary cells were then obtained by enzymatical digestion of TSGs and injected into the tibiae of mice to mimic a RCC bone metastasis. Mice were followed up with x-ray and micro-CT. At the end of the experiment the mice bearing intratibial tumors were sacrificed and the tibiae were fixed and longitudinally sectioned at 5-μm. The phenotype of the metastases and interactions of cancer cells with the bone marrow cells/stem cell niche was analyzed by means of immunohistology. Results: By using human specific qPCR, we detected disseminated tumor cells in bone of mice bearing TSGs derived from a clear cell carcinoma patient, who subsequently progressed to metastatic disease, 1 month after implantation. The analysis for 9 other RCC cases is currently under way. We then confirmed the presence of metastatic lesions in bone by histology. These metastases retained many phenotypic characteristics of the primary tumor (as shown by histology and immunohistochemistry for CA-IX or CD-31). Primary cells derived from the TSGs produced x-ray visible tumors/metastases already at 3 weeks after cancer cell inoculation intratibially at a 100 % engraftment rate. Micro-CT analysis revealed an osteolytic nature of these lesions, which is considered typical for renal cancer bone metastases. Conclusions: We showed that orthotopic RCC TSGs have potential to develop micrometastases and metastases to bone. We demonstrated the feasibility of an intratibial model of RCC metastasis to bone that resembles the actual disease in many ways. These are the first reported models of primary RCC metastasis to bone. Our translational research project allows realistic settings to test therapeutics to prevent or treat micro-metastases and metastases in RCC and has potential for applications in personalized medicine Citation Format: Maija Valta, Hongjuan Zhao, Alexandre Ingels, Alan Thong, Rosalie Nolley, Matthias Saar, Donna Peehl. Mouse model of primary human renal cell carcinoma metastasis to bone. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B9.

MIP-1δ Activates NFATc1 and Enhances Osteoclastogenesis: Involvement of Both PLCγ2 and NFκB Signaling

Pathological bone resorption is a source of significant morbidity in diseases affecting the skeleton such as rheumatoid arthritis, periodontitis, and cancer metastasis to bone. Evidence indicates that elevated levels of inflammatory mediators such as IL-1, IL-6, and TNF-α play a role in this process by promoting the formation of bone-resorbing osteoclasts. Additionally, current studies have identified inflammatory chemokines of the macrophage inflammatory protein (MIP) family as potential mediators of pathological bone resorption, where both MIP-1α and -3α have been shown to enhance osteoclast (OCL) development. In this study we provide evidence that MIP-1δ, whose expression is associated with renal cell carcinoma bone metastasis and rheumatoid arthritis, enhances OCL formation in vitro via a direct effect on OCL precursors. Consistent with this ability, exposure of OCL precursors to MIP-1δ resulted in the activation of PLCγ2 and NF-κB, two signaling pathways known to regulate OCL differentiation. Moreover, MIP-1δ induced expression and nuclear translocation of NFATc1, a master regulator of osteoclastogenesis, which was dependent on activation of both the PLCγ2 and NFκB signaling pathways. Lastly, consistent with in vitro studies, in vivo administration of MIP-1δ significantly increased OCL number and resorption area as determined using a murine calvarial bone resorption model. Taken together, these data highlight the potential of MIP-1δ as a mediator of pathological bone resorption and provide insight into the molecular mechanism through which MIP-1δ enhances osteoclastogenesis.

Abstract 1411: Calcium-depending formation of bone metastases in patients with renal cell carcinoma

Abstract Introduction: Although a number of molecules have been implicated in the process of formation of cancer metastases, the organ-selective nature of cancer cells is still poorly understood. Renal cell carcinoma (RCC) often metastasize into bones, a calcium enriched tissue. We analyzed the influence of the calcium sensing receptor (CaSR) in RCC and an enhanced extracellular calcium concentration in processes of metastatic spread. Material and Methods: In 33 matched RCC specimens, 11 non-metastasized, 11 metastasized into the lung and 11 metastasized into bones five years after nephrectomy, CaSR was quantified by RT-PCR and Western blot analyses. To investigate the influence of enhanced extracellular calcium on the formation of metastases, primary RCC cells were isolated from patients developing bone, lung and no metastasis five years after nephrectomy (each 3 cell lines). Cell proliferation after calcium treatment (2.5, 5 and 10 µM) was quantified by BrdU incorporation. Cell migration was determined in a Boyden chemotaxis assay with calcium as chemotactical agent (10 µM). Expression and activity of intracellular signal transduction mediators were analyzed by a human phospho kinase array and Western blot analyses. Results: Compared to non-metastasizing RCC, CaSR mRNA expression was 10.8-fold increased and CaSR protein level was 1.5-fold increased in tumor specimens of patients developing bone metastases. In primary RCC cells with a high bone metastastic potential CaSR expression was 3.7-fold higher compared to non-metastasizing RCC cells. Calcium treatment of bone-metastasizing RCC cells, but not of non-metastasizing cells, induced a concentration dependent enhancement of proliferation. In a Boyden chamber experiment with calcium as chemo attractant, RCC cells from patients with bone metastases showed a 9.4-fold higher migratory potential compared to cells from patients without metastases. In lung metastatic RCC cells migration was only 1.7-fold enhanced. The enhanced proliferation and migration of cells with high potential to build bone metastases seem to be caused by an activation of Akt and a highly increased integrin alpha 5 expression. Additionally, the tumor suppressor PTEN was markedly reduced in tumors metastasizing into bones and after calcium treatment, promoting tumor progression. Conclusion: Our results indicate that formation of bone metastases of RCC is caused by an enhanced expression of the CaSR. Extracellular calcium has a promoting effect on cell migration and proliferation of bone metastasizing RCC cells via Akt signalling and integrin alpha 5. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1411. doi:1538-7445.AM2012-1411