GeneTrail 3.2

Advanced high-throughput enrichment analysis

Wilms' tumors

The Wilms’ tumor or nephroblastoma is the most common malignant renal cancer in children, affecting one in 10,000 children [1]. Approximately 75% of the cases occur in children younger than five years old, with a peak incidence in two- to three- year-olds [2]. 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% [3]. Current therapeutic approaches rely on the classification of the tumor stage and histological subtype [4]. 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 [5] was used. This classification scheme is the standard for renal tumors of the childhood that underwent a preoperative chemotherapy.

Analysis

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.

Technical background

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) [6], which is equivalent to the standard Kolmogorov-Smirnov statistic [7]. 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 [8].

Parameter

  • 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

Step-by-step slideshow

The following slideshow depicts the different analysis steps of the GeneTrail2 workflow.

Results

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 [9]. 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 [10]. 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.

Generic placeholder image
Generic placeholder image

Immune response

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.

Generic placeholder image
Generic placeholder image
Generic placeholder image

Aggressive potential

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.

Generic placeholder image
Generic placeholder image

Wnt signaling

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 [11]. It has been reported to play a central role in Wilms’ tumors [12] and it has been identified as therapeutic target in other tumors [13].

Generic placeholder image

Summary

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 [14] [3].

Bibliography

  1. Maschietto, Mariana and Piccoli, Fabio S and Costa, Cecilia ML and Camargo, Luiz P and Neves, Jose I and Grundy, Paul E and Brentani, Helena and Soares, Fernando A and de Camargo, Beatriz and Carraro, Dirce M Gene expression analysis of blastemal component reveals genes associated with relapse mechanism in Wilms tumour European Journal of Cancer Elsevier
  2. Dangio, G. J. Wilms tumor status report, 1990 Journal of Clinical Oncology WB Saunders Co Independence Square West Curtis Center, STE 300, Philadelphia, PA 19106-3399
  3. Graf, Norbert and Tournade, Marie-France and de Kraker, Jan The Role of Preoperative Chemotherapy in the Management of Wilms Tumor: The SIOP Studies Urologic Clinics of North America Elsevier
  4. Green, Daniel M The treatment of stages I--IV favorable histology Wilms' tumor Journal of clinical oncology American Society of Clinical Oncology
  5. Vujanic, Gordan M and Sandstedt, Bengt and Harms, Dieter and Kelsey, Anna and Leuschner, Ivo and de Kraker, Jan Revised International Society of Paediatric Oncology (SIOP) working classification of renal tumors of childhood Medical and pediatric oncology Wiley Online Library
  6. Backes, Christina and Keller, Andreas and Kuentzer, Jan and Kneissl, Benny and Comtesse, Nicole and Elnakady, Yasser A and Müller, Rolf and Meese, Eckart and Lenhof, Hans-Peter GeneTrail—advanced gene set enrichment analysis Nucleic acids research Oxford Univ Press (View online)
  7. Hollander, Myles and Wolfe, Douglas A and Chicken, Eric Nonparametric statistical methods John Wiley and Sons
  8. Keller, A. and Backes, C. and Lenhof, H. P. Computation of significance scores of unweighted Gene Set Enrichment Analyses BMC Bioinformatics (View online)
  9. Kanehisa, Minoru and Goto, Susumu TNF signaling pathway — KEGG (hsa04668) (View online)
  10. Kanehisa, Minoru and Goto, Susumu NF-kappa B signaling pathway — KEGG (hsa04064) (View online)
  11. Kanehisa, Minoru and Goto, Susumu Wnt signaling pathway — KEGG (hsa04310) (View online)
  12. Major, Michael B and Camp, Nathan D and Berndt, Jason D and Yi, XianHua and Goldenberg, Seth J and Hubbert, Charlotte and Biechele, Travis L and Gingras, Anne-Claude and Zheng, Ning and MacCoss, Michael J and others Wilms tumor suppressor WTX negatively regulates WNT/beta-catenin signaling Science American Association for the Advancement of Science
  13. Anastas, Jamie N and Moon, Randall T WNT signalling pathways as therapeutic targets in cancer Nature Reviews Cancer Nature Publishing Group
  14. Graf, N and Weirich, A and de Kraker, J Staging problems in the pre-operative chemotherapy of Wilms' tumour. British journal of urology