No abstract

Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens.

Total RNA can be extracted from archival renal biopsies in sufficient quality and quantity from one human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits.

Clear cell renal cell carcinoma (ccRCC) represents the most common type of kidney cancer with high mortality in its advanced stages. Our study aim was to explore the correlation between tumor epithelial‐to‐mesenchymal transition (EMT) and patient survival. Renal biopsies of tumorous and adjacent nontumorous tissue were taken with a 16 g needle from our patients (n = 26) undergoing partial or radical nephrectomy due to ccRCC. RNA sequencing libraries were generated using Illumina TruSeq® Access library preparation protocol and TruSeq Small RNA library preparation kit. Next generation sequencing (NGS) was performed on Illumina HiSeq2500. Comparative analysis of matched sample pairs was done using the Bioconductor Limma/voom R‐package. Liquid chromatography‐tandem mass spectrometry and immunohistochemistry were applied to measure and visualize protein abundance. We detected an increased generic EMT transcript score in ccRCC. Gene expression analysis showed augmented abundance of AXL and MMP14, as well as down‐regulated expression of KL (klotho). Moreover, microRNA analyses demonstrated a positive expression correlation of miR‐34a and its targets MMP14 and AXL. Survival analysis based on a subset of genes from our list EMT‐related genes in a publicly available dataset showed that the EMT genes correlated with ccRCC patient survival. Several of these genes also play a known role in fibrosis. Accordingly, recently published classifiers of solid organ fibrosis correctly identified EMT‐affected tumor samples and were correlated with patient survival. EMT in ccRCC linked to fibrosis is associated with worse survival and may represent a target for novel therapeutic interventions.

Epidemiological studies support a strong link between organ fibrosis and epithelial cancers. Moreover, clinical and experimental investigations consistently indicate that these diseases intertwine and share strikingly overlapping features. As a deregulated response to injury occurring in all body tissues, fibrosis is characterized by activation of fibroblasts and immune cells, contributing to progressive deposition of extracellular matrix (ECM) and inflammation. Cancers are driven by genetic alterations resulting in dysregulated cell survival, proliferation and dissemination. However, non-cancerous components of tumour tissues including fibroblasts, inflammatory cells and ECM play key roles in oncogenesis and cancer progression by providing a pro-mutagenic environment where cancer cells can develop, favouring their survival, expansion and invasiveness. Additional commonalities of fibrosis and cancer are also represented by overproduction of growth factors, like transforming growth factor β, epithelial-to-mesenchymal transition, high oxidative stress, Hippo pathway dysfunctions and enhanced cellular senescence. Here, we review advances in the analysis of cellular and molecular mechanisms involved in the pathogenesis of both organ fibrosis and cancer, with particular reference to chronic kidney diseases and renal cell cancers. Most importantly, improved understanding of common features is contributing to the development of innovative treatment strategies targeting shared mechanisms.