Correlative Clinical And Experimental Research On The TKI-Treatment Of Advanced Renal Cell Carcinoma

Targeted therapy with tyrosine kinase inhibitor (TKI) improves clinical outcomes for patients with advanced renal cell cancer (RCC). However clinical and biological prognosis factors with more efficacy and accuracy are in short. Dose reduction and medication interruption caused by drug-related toxicity may increase the risk of treatment failure. In addition, precise and effective methods for treatment assessment and clinical condition monitoring are in need. Therefore, this study aimed to explore the prognosis factors based on the data of the advanced RCC patients treated with TKI in our single medical center, and to discuss the clinical significance of CAIX, HIF-2α, VEGFR-2 and VEGFR-3 expression in tumor tissue as prognosis factors for TKI therapy. By analyzing the relationship between plasma concentration of TKI and drug-related toxicity, we explore the potential clinical value of therapeutic drug monitoring for advanced RCC patients treated with TKI in outpatients. And furthermore we attempt to verify the feasibility of SET-iFISH technique circulating tumor cells (CTC) testing from peripheral blood and lay the foundation for application of CTC detection in the field of diagnosis and molecular targeted therapy for patients with RCC.Part Ⅰ:Clinical outcomes of TKI treatment for advanced renalcell carcinoma and testing of associated molecules markers Objective:Retrospectively analyze the clinical outcomes of advanced renal cell carcinoma patients treated with TKI, and evaluate the possible prognostic factors. And further discuss the value of CAIX, HIF-2a, VEGFR-2 and VEGFR-3 expression in tumor tissue as molecular markers for treatment response and prognosis.Materials and methods:Retrospective study was performed on the data of 242 cases of advanced renal cell carcinoma patients treated with TKI therapy. And progression-free survival (PFS) and overall survival (OS) were evaluated using the Kaplan-Meier method, and under Log-rank test. Univariate and multivariate Cox proportional hazards model were used to analyze the prognostic factors for 242 cases of advanced RCC patients treated with TKI. And CAIX, HIF-2α, VEGFR-2 and VEGFR-3 immunohistochemical analysis were performed in 57 cases of clear-cell renal clear cell carcinoma (ccRCC) tissue samples. And the immunohistochemical results were compared with the clinical response and prognosis.Results:The median PFS and OS were 11.4 and 24.2 months respectively and the objective response rate (ORR) and disease control rate (DCR) were 14.8% and 82.6%. Multivariate Cox proportional hazards model evaluation showed high ECOG score, lymph node metastasis and bone metastasis were independent risk factors for disease progression and overall survival for patients with TKI therapy. CAIX, HIF-2α, VEGFR-2 and VEGFR-3 expressed positively with different degrees in ccRCC. Patients with VEGFR-3 positive in ccRCC sample were more susceptible to lymph node metastasis (p = 0.0433). The difference between PFS and patients with different expression of the four proteins was not significant, neither was the efficacy response. Furman pathological grade was an independent risk factor of disease progression for patients with advanced RCC treated with TKI (HR:2.141,95% CI:1.222-3.752, p=0.0078).Conclusions:Targeted therapy with TKI improves clinical outcomes with varying degree for patients with advanced RCC. VEGFR-3 expression in ccRCC tissue may be related with lymph node metastasis in patients with RCC. However by testing the expression of CAIX, HIF-2a, VEGFR-2 and VEGFR-3 in ccRCC tissue can not be served as factors of prognosis and response for patients with TKI therapy. Patients’ prognosis is more likely predicted by physical performance, lymph node metastasis, bone metastasis and tumor grade. tissue samples. And the immunohistochemical results were compared with the clinical response and prognosis.Part Ⅱ:The association between TKI plasma concentration and drug-related toxicityObjective:To study the influence of sorafenib and sunitinib related toxicity on the treatment and prognosis in patients with advanced RCC. And further to explore the relationship between the plasma steady-state trough concentrations (Ctrough,ss) of drug and toxicity, and further to evaluate the potential clinical significance of therapeutic drug monitoring of TKI for patients with advanced RCC.Materials and methods:Influence of toxicity on TKI-treatment and prognosis were analyzed in 164 cases of sorafenib and 78 cases of sunitinib treatment patients. Ctrough,ss were tested for 32 cases of patients with sorafenib therapy (69 blood samples) and 23 cases of patients with sunitinib therapy (41 blood samples) by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). And the Ctrough,ss of sorafenib, sunitinib and its active metabolite (SU12662) were compared with drug related-toxicity.Results:There were 14.6% and 62.8% of patients with sorafenib and sunitinib therapy suffered from dose reduction due to toxicity, but the PFS and OS were not affected. Hypertension was a favorable factor of overall survival for patients with sorafenib therapy (HR:0.37,95% CI:0.16-0.84,p=0.0177). Individual difference of Ctrough,ss varied greatly in clinical patients with sorafenib and sunitinib therapy (CV=56.0%, CV= 56.4%). Plasma Ctrough,ss of sorafenib in patients presented with hand-foot syndrome (HFS)≥2 grade were higher than patients presented with HFS<1 grade, and plasma Ctrough,ss in patients presented with alopecia were higher than those without alopecia. The receiver operating characteristic curve (ROC) showed the cutoff of plasma sorafenib Ctrough, ss to predict HFS>2 and alopecia were 2669 ng/ml and 4589 ng/ml respectively. Plasma Ctrough, ss of sunitinib and total drugs in patients presented with drug-related hypertension were higher than those without hypertension. ROC showed the cutoff of sunitinib and total drugs Ctrough, ss to predict drug-related hypertension were 54.8 ng/ml and 73.7 ng/ml respectively.Conclusion:Dose reduction due to TKI related-toxicity does not attenuate the prognosis. Alopecia and the HFS are related to the plasma concentration of sorafenib, and hypertension is related to the plasma concentration of sunitinib. Individual difference of drug concentration varied greatly in clinical patients with TKI therapy. Therapeutic drug monitoring of TKI holds potential clinical value for therapy individualization, however more pharmacokinetic data is still needed. cases of patients with sunitinib therapy (41 blood samples) by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). And the Ctrough,ss of sorafenib, sunitinib and its active metabolite (SU12662) were compared with drug related-toxicity.Part Ⅲ:Result of circulating tumor cell testing from peripheral blood in patients with renal cell carcinoma by SET-iFISHObjective:To explore the feasibility of SET-iFISH method testing circulating tumor cell (CTC) in patients with renal cell carcinoma (RCC), and further lay the foundation of CTC testing for clinical application of diagnosis and molecular targeted therapy for patients with RCC.Materials and methods:9 cases of patients with RCC (2 cases of T4N1M0,1 case of T3cN0M0,1 case of T2aN0M0,1 case of T1bN0M0,3 cases of T1aN0M0 and 1case of TxN0M1) and 1 cases of renal calculus hospitalized in the urology department of our hospital were enrolled.7.5ml of peripheral blood samples were collected. Magnetic beads coupling with anti-leukocyte surface markers CD45 monoclonal antibody were combined to the leukocytes, and leukocytes were removed in the magnetic shelf. We chose fluorescent anti-cytokeratin (panCK:CK4,5,6,8,10,13 and 18) antibodies as CTC makers and combine with chromosome 8 fluorescence in situ hybridization (FISH) for quantitative detection of CTC in peripheral blood.Results:Quantitative test of CTC resulted in 6 cases of RCC patients with positive (2 cases of T4N1M0,1 case of T3cN0M0,2 cases of T1aN0M0 and 1 case of T1bN0M0),3 cases of RCC with negative (1 case of TxN0M1, T1aN0M0 and T2aN0M0) and 1 renal calculus with negative.3 cases of RCC were found in peripheral blood with circulating tumour microemboli (1 case of T1bN0M0, T4N1M0 and T1aN0M0). PanCK immunofluorescence staining for all samples were negative and the detection of CTC were based on chromosome 8 fluorescence in situ hybridization.Conclusion:SET-iFISH technique is capable of CTC detection for patients with different stages of RCC, and false positives is not presented temporarily. However the false negative is still beyond evaluated, thus an extended sample size and multiple testing are needed to verify the reliability of the method. CTC detection holds promising application prospects in the diagnosis and molecular targeted therapy for renal cell carcinoma, however a more accurate detection technology is required.