Limited utility of qPCR-based detection of tumor-specific circulating mRNAs in whole blood from clear cell renal cell carcinoma patients

Abstract

Background: RNA sequencing data is providing abundant information about the levels of dysregulation of genes in various tumors. These data, as well as data based on older microarray technologies have enabled the identification of many genes which are upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched normal tissue.Here we use RNA sequencing data in order to construct a panel of highly overexpressed genes in ccRCC so as to evaluate their RNA levels in whole blood and determine any diagnostic potential of these levels for renal cell carcinoma patients.Methods: A bioinformatics analysis with Python was performed using TCGA, GEO and other databases to identify genes which are upregulated in ccRCC while being absent in the blood of healthy individuals. Quantitative Real Time PCR (RT-qPCR) was subsequently used to measure the levels of candidate genes in whole blood (PAX gene) of 16 ccRCC patients versus 11 healthy individuals. PCR results were processed in qBase and GraphPadPrism and statistics was done with Mann-Whitney U test. Results: While most analyzed genes were either undetectable or did not show any dysregulated expression, two genes, CDK18 and CCND1, were paradoxically downregulated in the blood of ccRCC patients compared to healthy controls. Furthermore, LOX showed a tendency towards upregulation in metastatic ccRCC samples compared to nonmetastatic.Conclusions: This analysis illustrates the difficulty of detecting tumor regulated genes in blood and the possible influence of interference from expression in blood cells even for genes conditionally absent in normal blood. Testing in plasma samples indicated that tumor specific mRNAs were not detectable. While CDK18, CCND1 and LOX mRNAs might carry biomarker potential, this would require validation in an independent, larger patient cohort.

Identification of key genes and signalling pathways in clear cell renal cell carcinoma: An integrated bioinformatics approach

“…LOX expression is higher in the advanced stages of ccRCC [ 46 ]. Epithelial-mesenchymal transition (EMT), which is facilitated by LOX, encourages the invasion and metastasis of cancer [ 47 , 48 , 49 ]. HIF1 directly regulates the LOX expression and is found to be high in hypoxia-induced metastatic cancers [ 38 , 48 , 50 ].…”

Liquid Biopsies in Renal Cell Carcinoma—Recent Advances and Promising New Technologies for the Early Detection of Metastatic Disease

“…Recently, novel sequencing technologies have provided impetus to further investigate the potential of circulating mRNA and consequently CDK18 and CCND1 messengers were shown to be downregulated in blood of ccRCC patients (65) (Table 2). Similarly, an increase in lysyl oxidase (LOX) expression marked metastatic samples from the same cohort (65). In addition, circulating lncRNA (73), one of the newer players in the field of small non-coding RNAs, also showed promise as a RCC-specific biomarker.…”