Computational protocol: KCNJ3 is a new independent prognostic marker for estrogen receptor positive breast cancer patients

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Protocol publication

[…] The gene expression levels (RNAseq V2 level 3 data) of 950 invasive breast carcinoma samples and of 105 samples from corresponding healthy tissues were downloaded from the The Cancer Genome Atlas (TCGA) data portal ( and the upper quartile normalized counts from the RSEM pipeline were used. The corresponding clinical data of each patient were downloaded from the University of California Santa Cruz (UCSC) Cancer Genomics Browser ( Male patients (n = 9) and patients without gene expression data for KCNJ3 (n = 36) were excluded for further analysis (remaining n = 905). Patient characteristics are summarized in Table . [...] Slides were first assessed by microscopic inspection. Since the staining pattern of RNA in situ hybridization was homogenous across different regions of the same sample, a representative region was selected for each tumor, and z-stacks comprising 10 images were captured at 40× magnification using a Zeiss Observer.Z1 inverted microscope (Zeiss, Jena, Germany). Multiple adjacent single images (3x3 tiles) were acquired and aligned using the MosaiX module of the AxioVision software (Zeiss). Each assembled image covered an area of approx. 0.62 × 0.45 mm (3981x2980 pixels). Image sequences were stacked using the minimal intensity projection type setting of the ImageJ software ( The SpotStudio software from ACD was used for detection of single cells, detection of spots and clusters and calculation of the estimated number of spots per cell. DapB and POLR2A probes served as technical quality controls that needed to fulfill the below given cut-off criteria in order to a) ensure technical specificity of the probes (negative control) and b) to detect samples with highly degraded RNA (positive control). The maximum threshold for negative controls was set at 0.5 spots/cell and the minimum threshold for positive controls at 2.5 spots/cell. Samples not fulfilling these cut-off requirements were excluded from further analysis. [...] Statistical analyses were performed using the SigmaPlot/SigmaStat v12.5 software (Systat Software Inc., San Jose, CA, USA) or GraphPad Prism 7.02 for Windows (GraphPad Software, La Jolla, CA, USA) for comparison of groups, Spearman rank correlation analysis, generation of boxplots, bargraphs and scatter plots. All performed tests were two-sided and rank based. For comparisons of paired data, a Wilcoxon signed rank test, for comparisons of two groups a Wilcoxon rank sum test (Mann-Whitney U test), and for comparison for more than two groups a Kruskal-Wallis test followed by Dunn's posthoc tests for pairwise comparisons were used. Different patient characteristics between the TCGA cohort and the validation cohort (subset of GEO ID GSE17705) were compared using a chi-square test and a Wilcoxon rank sum test regarding age at diagnosis and follow-up times. Genesis 1.7.6 (Graz University of Technology, Graz, Austria) was used for log2-transformation and mean centering of gene expression values, calculation of Euclidean distance, hierarchical cluster analysis and heatmap visualization. For overall and disease free survival analysis, the statistical software environment R ( including the package survival and an adopted code for the auto-cut-off option from Györffy et al. [] was used. For the construction of survival curves, a Kaplan-Meier estimator was used and survival curves of patient groups with high expression versus low expression were compared by a logrank test. A time-independent Cox-proportional hazard approach was applied for univariate and multivariate survival analysis. Results were considered statistically significant when p < 0.05. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Databases TCGA Data Portal
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms
Chemicals Estrogens, Potassium