Øystein Eikrem

Transcriptome Sequencing (RNAseq) Enables Utilization of Formalin-Fixed, Paraffin-Embedded Biopsies with Clear Cell Renal Cell Carcinoma for Exploration of Disease Biology and Biomarker Development.

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.

RNA extraction for RNA sequencing of archival renal tissues.

Abstract Background: Next generation sequencing (NGS) and especially ribonucleic acid (RNA) sequencing is a powerful tool to acquire insights into molecular disease mechanisms. Therefore, it is of interest to optimize methods for RNA extraction from archival, formalin fixed and paraffin embedded (FFPE) tissues. This is challenging due to RNA degradation and chemical modifications. The aim of this study was to find the most appropriate method to extract RNA from FFPE renal tissue to enable NGS. Method: We evaluated seven commercially available RNA extraction kits: High Pure FFPE RNA Isolation (Roche), ExpressArt Clear FFPE RNAready (Amsbio), miRNeasy FFPE, RNeasy FFPE (Qiagen), PureLink FFPE Total RNA (Invitrogen), RecoverAll Total Nucleic Acid Isolation (Ambion) and Absolutely RNA FFPE Kit (Agilent). RNA was obtained from tissue blocks of two healthy, male Wistar rats and from normal renal tissue of patients undergoing nephrectomy. Yield and quality of RNA extracted from rat whole kidney sections, human kidney core biopsies and laser capture microdissected (LCM) glomerular cross-sections were assessed: Analyses of RNA quantity were performed using NanoDrop and Qubit. RNA quality is reflected by DV200 values (% of RNA fragments >200 nucleotides) utilizing the Agilent 2100 BioAnalyzer. RNA of human LCM samples was subsequently sequenced using the Illumina TruSeq® RNA Access Library Preparation Kit. Conclusion: 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.

Development and confirmation of potential gene classifiers of human clear cell renal cell carcinoma using next-generation RNA sequencing

Abstract Objective: A previous study by this group demonstrated the feasibility of RNA sequencing (RNAseq) technology for capturing disease biology of clear cell renal cell carcinoma (ccRCC), and presented initial results for carbonic anhydrase-9 (CA9) and tumor necrosis factor-α-induced protein-6 (TNFAIP6) as possible biomarkers of ccRCC (discovery set) [Eikrem et al. PLoS One 2016;11:e0149743]. To confirm these results, the previous study is expanded, and RNAseq data from additional matched ccRCC and normal renal biopsies are analyzed (confirmation set). Materials and methods: Two core biopsies from patients (n = 12) undergoing partial or full nephrectomy were obtained with a 16 g needle. RNA sequencing libraries were generated with the Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using linear modeling (voom/Limma; R Bioconductor). Results: The formalin-fixed and paraffin-embedded discovery and confirmation data yielded 8957 and 11,047 detected transcripts, respectively. The two data sets shared 1193 of differentially expressed genes with each other. The average expression and the log2-fold changes of differentially expressed transcripts in both data sets correlated, with R² = .95 and R² = .94, respectively. Among transcripts with the highest fold changes were CA9, neuronal pentraxin-2 and uromodulin. Epithelial–mesenchymal transition was highlighted by differential expression of, for example, transforming growth factor-β1 and delta-like ligand-4. The diagnostic accuracy of CA9 was 100% and 93.9% when using the discovery set as the training set and the confirmation data as the test set, and vice versa, respectively. These data further support TNFAIP6 as a novel biomarker of ccRCC. TNFAIP6 had combined accuracy of 98.5% in the two data sets. Conclusions: This study provides confirmatory data on the potential use of CA9 and TNFAIP6 as biomarkers of ccRCC. Thus, next-generation sequencing expands the clinical application of tissue analyses.

Pathomechanisms of renal Fabry disease

Fabry disease, also known as Anderson-Fabry disease, is a clinically heterogeneous, X-linked lysosomal storage disorder first described in 1898 by Johannes Fabry and William Anderson (Anderson 1898; Fabry 1898). The disease is caused by defects in the lysosomal enzyme, alphagalactosidase A (Brady et al. 1967; Sweeley and Klionsky 1963). Various mutations in the GLA-gene give rise to this rare form of lysosomal storage disease (Bishop et al. 1986; Germain 2010). Fabry disease is characterized by multi-organ involvement (e.g., skin, heart, kidneys, eyes, blood vessels and central/peripheral nervous system) with a wide variety of symptoms (Germain 2010; Wise et al. 1962) due to accum u l a t i o n o f g l y c o s p h i n g o l i p i d s , p r i m a r i l y globotriaosylceramide (abbreviated as Gb3 or GL-3), in several different cell types. Fabry disease is a slowly progressing systemic disorder characterized by a variable disease course in affected patients (Germain 2010). The initial disease process starts early (Vedder et al. 2006), yet most patients display no symptoms during infancy. Usually, clinical signs appear between 3 and 10 years of age (Hopkin et al. 2008) in classically affected males, with a later onset of symptoms in non-classical patients as well as in many female patients with classical mutation (Wilcox et al. 2008). Classical Fabry patients typically develop chronic kidney disease (CKD) culminating in end-stage renal disease (ESRD) before the fifth decade (Fogo et al. 2010). Enzyme replacement therapy (ERT), available for more than 15 years, has been shown to reduce GL-3 deposits and halt or attenuate progression of Fabry nephropathy in many patients. Disease progression is highly variable in females, ranging from nonprogressive asymptomatic carriers to classical Fabry disease. Clinically evident Fabry disease with kidney involvement, such as proteinuria and/or reduced glomerular filtration rate (GFR), beginning at an average age of 37 years has been reported in 40% of 248 investigated female patients registered in the Fabry Outcome Survey (Europe), including development of ESRD in three cases (Deegan et al. 2006). Male patients may present as early as in their second decade with evidence of chronic kidney disease (Branton et al. 2002a), while nephropathy was diagnosed at the mean age of 27 years in another study (Donati et al. 1987). In an NIH series, it was shown that 50 % of the male patients had chronic renal insufficiency at 43 years of age and half of the patients had reached ESRD 10 years later (Branton et al. 2002a). The renal aspects of Fabry disease contribute largely to the total burden of * Øystein Eikrem oystein.eikrem@uib.no

Clear Cell Renal Cell Carcinoma is linked to Epithelial‐to‐Mesenchymal Transition and to Fibrosis

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.

Bedside Stereomicroscopy of Fabry Kidney Biopsies: An Easily Available Method for Diagnosis and Assessment of Sphingolipid Deposits

Background/Aims: A previous case report found stereomicroscopic changes typical for Fabry disease in a kidney biopsy. This case series evaluates an expanded diagnostic capacity of the method. Methods: Bedside stereomicroscopy was performed in a cross-sectional prospective study of 31 consecutive enzyme-treated or treatment-naïve male (n = 14) and female Fabry disease patients. The burden of glomerular storage material was scored semiquantitatively on a visual analog scale (range 0-3) and a blinded comparison was done with a reference histologic method. Results: Significant correlations (p < 0.001) were found between the stereomicroscopic scoring of glomerular characteristic white storage material and the amount of podocyte globotriaosylceramide (Gb3) deposits scored by standardized light microscopy. The bedside method correctly identified the variability of podocyte Gb3 accumulation after 10 years of identical agalsidase therapy in 2 brothers aged 24 and 27 years, and also identified tubular cell deposits. Stereomicroscopy correctly verified the absence of sphingolipid deposits in the biopsy of a female index patient with a genetic variant of unknown significance, and the diagnosis of Fabry disease was finally discarded. Conclusions: Bedside stereomicroscopy of kidney biopsies is an easily available, low-cost microscopy method handled by the clinician. The method carries a high diagnostic sensitivity for Fabry disease, reducing the risk of misdiagnosis in previously unknown cases. An expanded yield of the method is suggested, including the grading of the podocyte Gb3 burden and assessment of effectiveness of enzyme replacement therapy. We recommend the method as complementary to current standard histologic evaluation of Fabry kidney biopsies.

Expanding the Utilization of Formalin-Fixed, Paraffin-Embedded Archives: Feasibility of miR-Seq for Disease Exploration and Biomarker Development from Biopsies with Clear Cell Renal Cell Carcinoma

Novel predictive tools for clear cell renal cell carcinoma (ccRCC) are urgently needed. MicroRNAs (miRNAs) have been increasingly investigated for their predictive value, and formalin-fixed paraffin-embedded biopsy archives may potentially be a valuable source of miRNA sequencing material, as they remain an underused resource. Core biopsies of both cancerous and adjacent normal tissues were obtained from patients (n = 12) undergoing nephrectomy. After small RNA-seq, several analyses were performed, including classifier evaluation, obesity-related inquiries, survival analysis using publicly available datasets, comparisons to the current literature and ingenuity pathway analyses. In a comparison of tumour vs. normal, 182 miRNAs were found with significant differential expression; miR-155 was of particular interest as it classified all ccRCC samples correctly and correlated well with tumour size (R2 = 0.83); miR-155 also predicted poor survival with hazard ratios of 2.58 and 1.81 in two different TCGA (The Cancer Genome Atlas) datasets in a univariate model. However, in a multivariate Cox regression analysis including age, sex, cancer stage and histological grade, miR-155 was not a statistically significant survival predictor. In conclusion, formalin-fixed paraffin-embedded biopsy tissues are a viable source of miRNA-sequencing material. Our results further support a role for miR-155 as a promising cancer classifier and potentially as a therapeutic target in ccRCC that merits further investigation.