Ryuichi Mizuno

Increased RANKL expression is related to tumour migration and metastasis of renal cell carcinomas

Receptor activator of NF‐κB ligand (RANKL) and its receptor, receptor activator of NF‐κB (RANK), play a key role in osteoclastogenesis, and osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We investigated the role of the RANKL–RANK–OPG system in renal cell carcinomas (RCCs), which frequently metastasize to bones. Real‐time quantitative PCR revealed that RANKL mRNA expression was higher in clear cell RCCs than in papillary and chromophobe RCCs. Similarly, RANKL protein expression level in clear cell RCCs was higher than that in papillary and chromophobe RCCs, showing positive correlations with the primary tumour stage and distant metastasis. There was no significant association between the expression level of RANK, OPG and histological subtypes of RCC. RANKL and RANK expression was observed in metastatic RCCs in the bone and other organs, suggesting that they play a role in metastasis to the bone and other organs. Recombinant RANKL protein stimulated migration of a clear cell RCC cell line, Caki‐1, in vitro, and this enhanced migration was inhibited by the administration of recombinant OPG protein. Furthermore, multivariate Cox analysis revealed that elevated RANKL and RANK expression with low‐OPG expression was a significant and independent predictor of recurrence, bone metastasis and a poor prognosis. These data suggest that the RANKL–RANK–OPG system is involved not only in the bone metastasis of RCCs but also in metastasis to other organs through the stimulation of cancer cell migration. Copyright

Expression of Snail and Slug in renal cell carcinoma: E-cadherin repressor Snail is associated with cancer invasion and prognosis

The Snail family transcription factors have been proposed as important mediators of epithelial–mesenchymal transition because of their role in down-regulation of E-cadherin and up-regulation of matrix metalloproteinases (MMPs). The present study was undertaken to investigate the expression of Snail, Slug and their associations with cancer invasion and prognosis in renal cell carcinomas (RCCs). Ninety-seven primary RCCs were analyzed for the protein expression of Snail, Slug, MMP2 and MMP9 by immunohistochemistry. Snail protein expression level was positively correlated with pathological tumor stage, histological grade and the presence of sarcomatoid carcinoma. On the contrary, Slug protein expression level was negatively correlated with pathological tumor stage, suggesting that Slug was down-regulated in advanced RCCs. Because Snail was positively associated with malignant potential of RCCs, involvement of Snail in the invasiveness of an RCC cell line 786-O was examined in the Matrigel invasion assay by down-regulating the gene expression with small interfering RNA (siRNA). Targeting the Snail, not Slug, expression in 786-O cells with siRNA caused down-regulation of the gene expression of Snail, vimentin, MMP2 and MMP9, but up-regulated the E-cadherin. Invasion of the cells through Matrigel in vitro was inhibited under this condition. Furthermore, expression levels of MMP2 and MMP9 were positively correlated with pathological tumor stage and the presence of sarcomatoid carcinoma. Statistical analysis indicated that elevated Snail, MMP2 and MMP9 protein expression are significantly worse predictors of disease-free and disease-specific survival of the patients with RCC. In conclusion, these data suggest that Snail has an important role in invasion and metastasis, and that silencing the gene may be a potential therapeutic target in RCCs.

Expression of TNF-α and CD44 is implicated in poor prognosis, cancer cell invasion, metastasis and resistance to the sunitinib treatment in clear cell renal cell carcinomas

Tumor necrosis factor‐α (TNF‐α) is involved in epithelial‐mesenchymal transition (EMT) and expression of CD44, a cancer stem cell marker, in several cancers. This study was performed to clarify the significance of TNF‐α and CD44 in clear cell renal cell carcinomas (ccRCCs). Expression of TNF‐α and CD44 was examined by immunohistochemistry in 120 ccRCCs. Involvement of TNF‐α in EMT and induction of CD44 was analyzed by monitoring expression of EMT‐related genes and CD44, and invasion in cultured ccRCC cell lines. TNF‐α and CD44 were immunolocalized mainly to carcinoma cells of high‐grade ccRCCs with positive correlations with primary tumor stage. A positive correlation was also obtained between TNF‐α and CD44 expression, and co‐upregulation of TNF‐α and CD44 was associated with primary tumor stage, distant metastasis, and poor prognosis. TNF‐α enhanced migration and invasion of ccRCC cells together with down‐regulation of E‐cadherin expression and up‐regulation of matrix metalloproteinase 9 and CD44 expression. TNF‐α also up‐regulated the expression of TNF‐α itself in ccRCC cells. Among the 25 ccRCC patients treated with sunitinib for metastatic disease, high CD44 expression was associated with poor treatment outcome. Importantly, residual carcinoma cells in the sunitinib‐treated metastatic ccRCCs were strongly positive for CD44, and the CD44 expression was significantly higher in the tumors from the sunitinib‐treated patients than in those from untreated ones. Our data show that TNF‐α plays an important role in progression of ccRCCs by inducing EMT and CD44 expression, and suggest that CD44 induced by TNF‐α may be involved in the resistance to the sunitinib treatment.

Decreased Acetylation of Histone H3 in Renal Cell Carcinoma: A Potential Target of Histone Deacetylase Inhibitors

PURPOSE Recent studies suggest that alterations in chromatin structure by histone acetylation may have an important role in the neoplastic process. We evaluated histone H3 acetylation in renal cell carcinoma and the effects of a histone deacetylase inhibitor on renal cancer cell lines. MATERIALS AND METHODS The expression of acetylated histone H3 (Lys9) in renal cell carcinoma and corresponding nonneoplastic tissue specimens was evaluated. We next assessed immunohistologically the relationships between acetylated histone H3 expression and clinicopathological parameters in 44 cases of renal cell carcinoma. Furthermore, we evaluated the effects of the histone deacetylase inhibitor depsipeptide on the renal cancer cell lines Caki-1, ACHN, 769P and 786O (ATCC). RESULTS Acetylated histone H3 expression was decreased in 85.0% of renal cell carcinomas compared to levels in nonneoplastic tissue. Decreased expression was related to high nuclear tumor grade and advanced pathological tumor stage (p = 0.048 and 0.032, respectively). Depsipeptide inhibited cell line proliferation in a time and dose dependent manner. Depsipeptide induced apoptosis in Caki-1, ACHN and 786O, and induced G2 cell cycle arrest in 769P. Concomitantly depsipeptide increased acetylated histone H3 and p21(WAF/CIP1) expression, and induced Bcl-2 phosphorylation. CONCLUSIONS These results suggest that the decreased acetylation of histone H3 is a common alteration in a malignant phenotype of renal cell carcinoma. Increasing the amount of acetylated histone H3 might be a therapeutic option for renal cell carcinoma.

Incidence of seed migration to the chest, abdomen, and pelvis after transperineal interstitial prostate brachytherapy with loose 125 I seeds

BackgroundThe aim was to determine the incidence of seed migration not only to the chest, but also to the abdomen and pelvis after transperineal interstitial prostate brachytherapy with loose 125I seeds.MethodsWe reviewed the records of 267 patients who underwent prostate brachytherapy with loose 125I seeds. After seed implantation, orthogonal chest radiographs, an abdominal radiograph, and a pelvic radiograph were undertaken routinely to document the occurrence and sites of seed migration. The incidence of seed migration to the chest, abdomen, and pelvis was calculated. All patients who had seed migration to the abdomen and pelvis subsequently underwent a computed tomography scan to identify the exact location of the migrated seeds. Postimplant dosimetric analysis was undertaken, and dosimetric results were compared between patients with and without seed migration.ResultsA total of 19,236 seeds were implanted in 267 patients. Overall, 91 of 19,236 (0.47%) seeds migrated in 66 of 267 (24.7%) patients. Sixty-nine (0.36%) seeds migrated to the chest in 54 (20.2%) patients. Seven (0.036%) seeds migrated to the abdomen in six (2.2%) patients. Fifteen (0.078%) seeds migrated to the pelvis in 15 (5.6%) patients. Seed migration occurred predominantly within two weeks after seed implantation. None of the 66 patients had symptoms related to the migrated seeds. Postimplant prostate D90 was not significantly different between patients with and without seed migration.ConclusionWe showed the incidence of seed migration to the chest, abdomen and pelvis. Seed migration did not have a significant effect on postimplant prostate D90.

Preoperative Prognostic Nomogram (Probability Table) for Renal Cell Carcinoma Based on TNM Classification

PURPOSE Recently several prognostic nomograms have been developed to predict the prognosis of malignant diseases, including renal cell carcinoma. However, to our knowledge a preoperative prognostic nomogram that predicts survival in patients with renal cell carcinoma is not available. We developed a preoperative nomogram based on the TNM classification that predicts cause specific survival in patients with renal cell carcinoma. MATERIALS AND METHODS A total of 545 patients with renal cell carcinoma, including metastatic disease, who underwent radical nephrectomy or nephron sparing surgery at our institution were included in the study. Cases were staged according to the 2002 UICC TNM system, 6th edition. T, N and M factors were used as prognostic factors and a Cox proportional hazards regression model was developed to predict cause specific survival. A nomogram to predict cause specific survival was developed by repeating the analysis on 200 bootstrap samples. To validate the nomogram a concordance index was estimated and calibration was also examined by plotting the predictions made by the nomogram. RESULTS Overall 1, 3 and 5-year patient survival was 95.2%, 92.0% and 89.9%, respectively. T, N and M factors were significant prognostic factors in the Cox proportional hazards regression model. Using the combined TNM factors we developed a nomogram predicting 1, 3 and 5-year cause specific survival rates. The nomogram had excellent ability to discriminate, as evidenced by a concordance index of 0.81, and it was generally well calibrated. CONCLUSIONS The preoperative information shown by this nomogram may be important for obtaining informed consent from patients with renal cell carcinoma who have indications for surgery.

Expression of heparanase in renal cell carcinomas: Implications for tumor invasion and prognosis

Purpose: Heparanase activity has been detected in many malignant tumors, showing a correlation with the metastatic potential. The present study was undertaken to investigate the expression of heparanase and its prognostic significance in renal cell carcinomas (RCC). Experimental Design: Nineteen RCCs and 6 nonneoplastic renal tissues were analyzed for heparanase mRNA expression by real-time PCR. Heparanase protein expression was semiquantitatively investigated by immunohistochemistry in 70 RCCs. Involvement of heparanase in the invasiveness of RCC cell lines, 786-O and Caki-2 cells, was examined by down-regulating the gene expression with small interfering RNA (siRNA) using the Matrigel invasion assay. Results: The expression level of heparanase mRNA was significantly higher in clear cell RCCs than in papillary RCCs, chromophobe RCCs, and nonneoplastic renal tissues. Heparanase was predominantly immunolocalized to cell surface and cytoplasm of clear cell RCCs and mean expression levels of heparanase were significantly higher in clear cell RCCs than in papillary and chromophobe RCCs. The protein expression levels were positively correlated with primary tumor stage, distant metastasis, and histologic grade. Targeting of heparanase mRNA expression in 786-O and Caki-2 cells with siRNA down-regulated the mRNA expression and inhibited the Matrigel invasion by these cells, whereas nonsilencing siRNA showed no effect. Multivariate Cox analysis revealed that elevated heparanase expression was a significant and an independent predictor of disease-specific survival (odds ratio, 8.814; P = 0.019). Conclusions: These data suggest that heparanase plays an important role in invasion and metastasis and silencing of the gene might be a potential therapeutic target in clear cell RCCs.

Expression of Ets-1 in human clear cell renal cell carcinomas: implications for angiogenesis.

Expression of vascular endothelial growth factor (VEGF) has been reported in renal cell carcinoma (RCC), a highly angiogenic carcinoma. However, little or no information is available on the expression of Ets‐1, which is one of the target molecules of VEGF. In the present study, we examined the expression of Ets‐1 and VEGF in RCC by immunohistochemistry and reverse transcription–polymerase chain reaction (RT‐PCR), and correlations between expression and the microvessel density (MVD) were evaluated. Ets‐1 was immunolocalized to carcinoma cells and endothelial cells of the microvessels in clear cell RCC, but not in papillary RCC. Immunohistochemical Ets‐1 expression and MVD were significantly higher in clear cell RCC than in papillary RCC. Predominant mRNA expression of Ets‐1 in clear cell RCC was confirmed by RT‐PCR. The expression of Ets‐1 correlated directly with MVD in clear cell RCC. Hypoxic treatment upregulated the mRNA expression of Ets‐1 and VEGF in cell lines derived from clear cell RCC, suggesting that hypoxia is a key regulator for these molecules. These results demonstrate the expression of Ets‐1 in human clear cell RCC and suggest the possibility that Ets‐1 is involved in angiogenesis in clear cell RCC. (Cancer Sci 2006; 97: 875–882)

Epithelioid angiomyolipoma of the kidney: Radiological imaging

To review the imaging findings of renal epithelioid angiomyolipomas.

Patterns of Interstitial Lung Disease During Everolimus Treatment in Patients with Metastatic Renal Cell Carcinoma

OBJECTIVE To elucidate the patterns of interstitial lung disease during everolimus treatment in patients with metastatic renal cell carcinoma, we reviewed seven cases of everolimus-induced interstitial lung disease. METHODS Seven patients with metastatic renal cell carcinoma, which continued to progress despite treatment with sunitinib or sorafenib, developed interstitial lung disease after treatment with everolimus. RESULTS Chest X-ray demonstrated diffuse infiltrates in lung fields, and chest computed tomography showed bilateral reticular and ground-glass opacities. Serum levels of lactate dehydrogenase (7/7), C-reactive protein (6/7), pulmonary surfactant associated protein D (1/7) and Krebs von den Lungen 6 (5/7) were elevated. The bronchoalveolar lavage fluid obtained from four patients with Grade 3 interstitial lung disease showed lymphocytosis. The transbronchial lung biopsy specimens showed interstitial lymphocytic infiltration and septal thickening of alveolar walls. In two cases with mild interstitial lung disease, the everolimus therapy was successfully continued. In four cases with Grade 3 interstitial lung disease, the drug was discontinued and steroid therapy was initiated. Pulmonary symptoms and radiological abnormalities resolved within 2 months. CONCLUSIONS Serum Krebs von den Lungen 6 was elevated compared with baseline in all cases with interstitial lung disease. Some patients who developed mild interstitial lung disease during everolimus treatment could continue to receive the treatment. Even when severe interstitial lung disease developed, withdrawal of the drug and short-term use of high-dose steroids resulted in rapid recovery. Prompt recognition of interstitial lung disease exacerbation as well as exclusion of progressive disease or infection is of primary importance.