Clinical Study Of The Metastasis Of Urothelial Carcinoma And Related MicroRNAs

Part 1 Clinicopathological features and prognosis-associated factors of metastatic urothelial carcinoma patients【Objective】To investigate the clinicopathological features in metastatic urothelial carcinoma (MUC) patients, and evaluate overall survival (OS) associated factors.[Methods]We retrospectively reviewed 153 metastatic urothelial carcinoma (MUC) patients consecutively registered at peking union medical college hospital from 1997 January to 2015 February, and analyzed the data from three sapects.1. There were 71 patients with primary upper tract urothelial carcinoma (UTUC) and 82 with primary urothelial bladder cancer (UBC). We compared the characteristics of primary tumors, sites distribution of metastases and metastasis-free interval (MFI) between UTUC and UBC, and evaluated the clinicopathological factors associated with MFI.2. We investigated the prognosis asssociated factors in 50 MUC patients undergoing at least 2 cycles of chemotherapy, with complete follow-up data.3. We summarized the clinicopathological features of 51 urothelial carcinoma patients with bone metastasis, and evaluate the prognostic factors.【Results】1. There was no significant difference in the sites distribution and number of visceral metastases between UTUC and UBC. The most common site of metastases for both groups was lymph node, followed by lung, bone, liver and peritoneum. However, the primary tumors of metastatic UTUC were more invasive and the MFI of UTUC was significantly shorter (5.8 months vs.17.4 months,p<0.001). Univariate analysis revealed pathological stage was the only factor associated with the MFI of UTUC. Multivariate analysis showed that primary tumor grade was an independent inferior predictor for the MFI of UBC (HR 6.384,95% CI 3.21-12.69,p＜0.001), while recurrence was an independent favorable factor for the MFI of UBC (HR 0.384,95% CI 0.18-0.82, p=0.014).2. In our cohort, the median overall survival from the initiation of chemotherapy was 17.2 months. The overall survival rate was 68.0% at 1 year, and 24.0% at 2 years. In univariate analysis, visceral metastasis (p=0.032), bone metastasis (p=0.005), metastatic pattern (p=0.024), number of metastatic viscera (p=0.032), ECOG score (p＜0.001), primary tumor resection (p<0.001) and chemotherapy cycles (p<0.001) were associated with the prognosis of metastatic UC patients. Multivariate analysis indentified 3 clinical factors associated with worse prognosis:bone metastasis (HR 2.384,95% CI 1.178～4.824, p=0.016), ECOG score≥2 (HR 5.488,95% CI2.249～13.395,p＜0.001), four or less chemotherapy cycles (HR 2.498,95%CI 1.033～6.040, p=0.042).3. The most common sites of bone metastases in UC patients were pelvis, ribs, lumbar and thoracic vertebrae. The median bone metastasis free interval of UC from initial treatment was 13.0 months. The median OS from bone metastasis was 8.0 months, and the 1-year and 2-year OS rates were 31.4% and 9.8%, respectively. In univariate analysis, bone metastatic pattern (p=0.018), primary tumor resection (p=0.010), ECOG score (p=0.001) and chemotherapy (p=0.028) were associated with OS in bone metastatic UC patients. In multivariate analysis, synchronous bone metastasis (HR 2.558,95% CI 1.176-5.556,p=0.018) and ECOG score≥2 (HR 3.030,95% CI 1.536-5.988,p=0.001) were identified as independent adverse prognostic factors.【Conclusions】1. UTUC and UBC have the similar tissue tropism, but the primary tumors of metastatic UTUC were more invasive and the MFI of UTUC was significantly shorter compared to UBC. Retroperitoneal lymph node dissection should be advocated in UTUC patients who receive radical operations. Pathological stage was the only factor associated with the MFI in UTUC, while primary tumor grade and recurrence were independent factors for the MFI of UBC.2. Bone metastasis, ECOG score≥2 and four or less chemotherapy cycles were inferior prognostic factors in metastatic urothelial carcinoma patients undergoing at least 2 cycles of chemotherapy.3. Synchronous bone metastasis and ECOG score≥2 had independent negative influence on the survival time in UC patients with bone metastasis.Part 2 Preliminary study on metastasis associated microRNAs in urothelial carcinoma patients【Objective】To find out the difference of microRNA expression profiles between metastases and primary tumors in urothelial carcinoma, investigate the biological function of miR-146b-5p in metastasis process, and explore the potential molecular mechanisms.[Methods]1. Using a microarray platform which contains 3100 human microRNAs, we studied micorRNA profiles in paired primary tumors and metastases from 3 patients. Then, the relative expression levels of significant upregulated miR-146b-5p were analyzed in 22 formalin fixed and paraffin embedded samples and bladder cancer cell lines (5637 and T24) by quantitative real-time polymerase chain reaction (qRT-PCR).2. We stably overexpressed miR-146b-5p in two bladder cancer cell lines 5637 and T24 with lentiviruses carrying miR-146b-5p and its control miR-NC. The efficacy of infection was tested by qRT-PCR. Cell migration and invasion capacities were evaluated using woud healing test and transwell assay, respectively.3. The expression of E-cadherin and vimentin in 5637 and T24 cells were detected by western blot before and after transfection.【Results】1. As compared with primary tumors, we identified 51 microRNAs dysregulated in the metastases of urothelial carcinoma (fold change>2.0 andp<0.05), among which 32 were upregulated and 19 were downregulated. Notably, miR-146b-5p was remarkably upregulated with a fold change of 6.3. In accordance with microarray results, qRT-PCR showed that miR-146b-5p was upregulated in 22 formalin fixed and paraffin embedded metastases, and the expression level of miR-146b-5p in both 5637 and T24 was medium.2. miR-141 expression was elevated up to 18042-and 61-fold in 5637 and T24 cells, respectively. Woud healing test showed that overexpression of miR-146b-5p enhanced the migration of 5637 cells. Transwell assay revealed overexpression of miR-146b-5p significantly impaired invasiveness, both in 5637 and T24 cells.3. The expression of vimentin was not detected in 5637 cells, while the expression of E-cadherin was not detected in T24 cells. After transfection of miR-146b-5p, the expression of E-cadherin was upregulated in 5637 cells, and the expression of vimentin was downregulated in T24 cells.【Conclusions】 The microRNA expression profiles were different between the primary tumors and metastases in urothelial carcinoma. miR-146b-5p, significantly upregulated in metastases, enhances the migration of bladder cancer celss and reverses epithelial-mesenchymal transition. Thus, it may function as metastasis promoter in the process of organ adhesion and colonization in urothelial carcinoma.