Significance of gene expression analysis of renal cell carcinoma

533 Background: Sunitinib is a standard first line treatment of metastatic renal cell carcinoma (ccRCC). Approximately 20% of patients experience primary resistance to therapy and no predictive biomarkers are available. The aim of our study is to discover novel biomarkers to predict response to sunitinib and to generate hypothesis about mechanisms of intrinsic resistance. Methods: Gene expression analysis was performed in formalin-fixed paraffin embedded (FFPE) samples from 44 patients with metastatic ccRCC treated with sunitinib in our institution. Affymetrix Human Gene 2.0 ST array was performed in primary tumors from 6 extremely sensitive (progression free survival (PFS) > 24 months) and 8 refractory (progression disease as best response) ccRCC. Technical validation with qPCR (Fluidigm Dynamic 96.96 Array) was performed in the whole cohort. The ΔΔCt method was used to quantify the relative amount of mRNA. Clinical and pathological data were correlated with gene expression. Results: 330 genes were differentially expressed between refractory and sensitive patients (p≤0.05). Network analysis showed 16 significant networks represented. Gene expression of 96 selected markers was tested in 47 primary tumors and 24 metastases. We found IL8, VEGF and NOTCH pathways upregulated on refractory patients. Survival analysis showed that the overexpression of IL8 correlated with a worse PFS (HR: 2.42, 95%CI1.27 – 4.63, p=0.0075) and overall survival (OS) (HR: 3.42, 95%CI 1.50 – 7.79, p=0.0034). Overexpression of VEGFBcorrelated with a prolonged PFS (HR 0.52, 95% CI 0.28 – 0.98, p=0.0415). Conclusions: Our results confirm the predictive value of IL8 expression in a cohort of ccRCC patients treated with sunitinib and suggests novel biomarkers of response to sunitinib. These data will be further validated in an independent cohort of patients.

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Identification of Appropriate Housekeeping Genes for Gene Expression Studies in Human Renal Cell Carcinoma Under Hypoxic Conditions

10.21203/rs.3.rs-706573/v1 ◽

2021 ◽

Author(s):

Luiz Felipe S. Teixeira ◽

Rodrigo Gigliotti ◽

Luana da Silva Ferreira ◽

MARIA Helena Bellini

Keyword(s):

Gene Expression ◽

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Expression Analysis ◽

Renal Cell ◽

Gene Expression Analysis ◽

Housekeeping Genes ◽

Human Renal Cell Carcinoma ◽

Von Hippel Lindau ◽

Hypoxic Conditions

Abstract Background: Due to the loss of von Hippel-Lindau tumor suppressor function, clear renal cell carcinoma (ccRCC) deregulates hypoxia pathways. Quantitative PCR is a powerful tool for quantifying differential expression between normal and cancer cells. Reliable gene expression analysis requires the use of genes encoding housekeeping genes. Therefore, in this study, eight reference candidate genes were evaluated to determine their stability in 786-0 cells under normoxic and hypoxic conditions. Methods and Results: Four different tools were used to rank the most stable genes: GeNorm, NormFinder, BestKeeper, and Comparative Ct (ΔCt), and a general ranking was performed using the RankAggreg. According to the four algorithms, the TFRC reference gene was identified as the most stable, and therefore, no agreement was observed for the 2nd and 3rd positions. A general classification was then established using the RankAggreg tool. Finally, the three most suitable reference genes to be used in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA. Conclusions: To our knowledge, this is the first study to evaluate reliable genes that can be used in gene expression analysis in ccRcc under a hypoxic environment.

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