The Wilms’ tumor or nephroblastoma is the most common malignant renal cancer in children, affecting one in 10,000 children . Approximately 75% of the cases occur in children younger than five years old, with a peak incidence in two- to three- year-olds . Until recently, this tumor was always considered lethal, but serves today as a prime example of a curable malignant disease with survival rates of 90% . Current therapeutic approaches rely on the classification of the tumor stage and histological subtype . Identifying and understanding the different subtypes is crucial for finding a proper therapy and to assess the degree of malignancy of the tumor and thereby its aggressive potential. In order to describe the histology of the analyzed tumors the SIOP-classification scheme  was used. This classification scheme is the standard for renal tumors of the childhood that underwent a preoperative chemotherapy.
In this use case we compare gene expression profiles of regressive Wilms’ tumors, which show a positive response to the chemotherapy, to the blastemal subtype, which seems to have a stronger chemoresistance. We use GeneTrail2 to detect biological processes and molecular functions that show significant differences between the two groups.
As both groups only contain a few samples we decided to use the Shrinkage-t-test to compute the differences in gene expression between them (compare Workflow, Implemented methods). To perform the enrichment analysis we decided to use the unweighted version of the gene set enrichment analysis (GSEA) , which is equivalent to the standard Kolmogorov-Smirnov statistic . It is a non-parametric hypothesis test, which is based solely on the order of an input list L. Focusing on ranks rather than on the absolute value has the advantage that the method is more robust and can penalize outliers, which might otherwise have a big influence on the results. (compare Workflow, Implemented methods). Another advantage of the unweighted GSEA is that an exact p-value for the test statistic can be computed via a dynamic programming algorithm .
- Test set: Ordered list of expressed mRNAs
- Identifier-level statistics: Shrinkage t-test
- Set-level statistics: Gene set enrichment analysis (GSEA)
- P-value adjustment method: Benjamini-Hochberg
The following slideshow depicts the different analysis steps of the GeneTrail2 workflow.
In the following section, we describe the results of our enrichment analysis and try to find evidence for the stronger chemoresistance of the blastemal subtype and the aggressive potential of these tumor cells.
Impact of chemotherapy
By analyzing the enrichment results, we see a high activity of apoptotic processes and pathways that are involved in tumor necrosis in the regressive tissue. We see a high activity of the TNF signaling pathway, which induces apoptosis and inflammation as well as immunity . Additionally, the NF-kappa B signaling pathway is significantly enriched. This pathway contains many transcription factors that regulate genes involved in immunity, inflammation, and cell survival . This effect emerges from the high content of necrotic cells in this tissue and is a good indicator for the effectiveness of the chemotherapy in this group of patients.
The high necrotic content also seems to have a strong impact on the immune system in the regressive tissue. We see a high activity of the phagosome, chemokines, natural killer cells and T-cells.
Furthermore, we detect a significant down-regulation of many categories involved in the proliferation of cells in the regressive tissue. We see a lower activity of the cell cycle, ribosome, spliceosome, and DNA replication. This might be an indicator that the blastemal cells that survived the chemotherapy have a more aggressive potential and might lead to a more malignant progression of the disease.
Additionally, the enrichment analyses show a down-regulation of the Wnt signaling pathway. Wnt proteins are secreted morphogens that are required for basic developmental processes, such as cell-fate determination, progenitor-cell proliferation, and the control of asymmetric cell division in many different species and organs . It has been reported to play a central role in Wilms’ tumors  and it has been identified as therapeutic target in other tumors .
Altogether, we see that the chemotherapy seems to have different impacts on regressive and blastemal cells. The differences in gene expression clearly reflect the phenotype of the different tissues and reinforce the assumption that a high content of blastemal cells after chemotherapy might lead to a more unfavorable prognosis for the progression of the disease  .
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- TNF signaling pathway — KEGG (hsa04668) (View online)
- NF-kappa B signaling pathway — KEGG (hsa04064) (View online)
- Wnt signaling pathway — KEGG (hsa04310) (View online)
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