Axel Heidenreich - assoc. RG

Molecular mechanisms involved in migration, invasion and metastastic processes of testicular germ cell tumors

Summary

The aim of the Center for Molecular Medicine is to support state of the art basic as well as translational research in molecular biology in order to gain insight into the pathogenetic mechanisms of diseases and to develop new and more efficient therapeutic modalities. With its 41 research groups from the Faculty of Medicine and the Faculty of Natural Science the CMMC focuses on three main areas of research: Cardiac and Vascular Disorder, Host Immune Response to

Introduction

As with any given cancer TGCT consist of different cellular subpopulations with different abilities of growth, differentiation, metastatic potential, and drug resistance. TGCT are genetically heterogeneous and subpopulations with high metastatic potential may be present only in low quantities. The identification of specific biomarkers of such highly aggressive subpopulations represents a crucial issue in order to develop an individualized and risk adapted therapeutic approach in clinical stage I disease. Since the invasion front of cancer seems to play a specific role in tumor progression the isolation and molecular analysis of this particular tumor subpopulation appears to be the most appropriate approach to detect early molecular events involved in the metastatic process.

It is the purpose of this project to elucidate potential molecular events with differential expression profiles at the invasive front and tumor center in stage I testis cancer correlating these findings with the presence or development of metastases in clinical stage I TGCT. For the first time, this approach is used in testis cancer research respecting intratumoral heterogeneity of the cancer cells.

The objective of this project is to elucidate potential molecular events with differential expression profiles at the invasive front and tumor center in stage I testis cancer correlating these findings with the presence of metastases in clinical stage II and III TGCT.

Study design

Paraffin-embedded orchiectomy specimens of patients with clinical stage I seminomas (n=50) and clinical stage I nonseminomatous testicular germ cell tumors (NSGCT, n = 50) will be identified from our data registry of testis cancer patients. Only patients who have undergone active surveillance as first line therapy will be included. Patients with metastatic TGCT (n = 50) serve as a control group. 

Testis cancer cells located in the centre of the tumor, at the invasion front and intravascular tumor cells will be laser microdissected. Tissue specimens of the same patient without cancer or intraeoithelial neoplasia serve as negative controls. Total mRNA will be extracted from the microdissected cancer cells for further expression analysis including a panel 740 genes. The expression profile of these genes is analysed using the PanCancer Progression Panels untersucht. (Nanostring, Santa Clara, USA) which represent an mRRNA – based, digitalized and color coded barcode hybridization technique. Using this approach, the most relevant pathways involved in neoangiogenesis, extracellular matrix remodeling (ECM), epithelial-mesenchymal-transition (EMT) and metastazation can be identified. 

To analyse the small intravascular cancer cell clones, SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian (Takara Bio USA, Mountain View, CA, USA) will be used, which only needs 250pg – 10ng total-RNA to develop a cDNA libary for further sequencing analysis.

Genes with statistically significant different expression profiles will be further evaluated via immunohisochemical expression studies. 

Based on the results of our study, prospective, multicenter evaluation in a larger cohort of patients is planned. 

Preliminary results

Intratumoral Heterogeneity
Hierarchical cluster analysis showed no differential gene clustering between the metastatic and non-metastatic patients. Comparing TF and TC in the metastasized group more genes were significantly differentially expressed (log2 fold change >1.5, p-value <0.05) compared to non-metastasized patients. Pairwise comparison of TF and TC for each individual patient shows differential expression of genes in these two regions. Lasso regression analysis of TF revealed a signature of six genes that could predict metastasis with a specificity of 100%, and a sensitivity of 92.9%. However, with respect to the TC no similar gene signatures were identified. This study describes tumor heterogeneity in seminomas and identifies a gene signature that could predict metastasis and has to be validated on occult metastasized seminomatous patients.

Comprehensive analysis of metastatic seminoma germ cell tumors shows divergent expression of immune related pathways
Differential gene expression patterns were observed in the metastatic and non-metastatic patients, with respect to both the tumor front and tumor center regions. Ingenuity pathway analysis on the differentially expressed genes showed enrichment of tumor functions like migration, invasion, and angiogenesis at the TF as compared to the TC. Several immune related pathways (IL-6 Signaling, Acute Phase Response Signaling, NF-?B Signaling, Dendritic Cell Maturation) were significantly upregulated in the metastatic versus non-metastatic patients. This is the first study showing tumor heterogeneity in TGCTs. Evidently, IL-6 signalling was the most significantly upregulated pathway in the metastatic versus the non-metastatic patients, that could serve as a therapeutic target for personalized therapy.

Perspectives

For the very first time we have demonstrated that there is a significant intratumoral heterogeneity among patients with metastatic seminomas. Based on the results of 6 differentially expressed genes in metastatic versus nonmetastatic and relapse-free patients, new prognostic markers to initiating adjuvant treatment regimes might be integrated in the decision making process. Currently, the identified genes are validated in an external cohort of patients. In addition, we have identified upregulated IL-6 signalling pathways in metastatic seminomas. These data might serve as a basis for immune modulatory treatment approaches.


Prof. Dr. Axel Heidenreich

Dept. of Urology / RG location - LFI Building

Prof. Dr. Axel Heidenreich

assoc. CMMC Research Group

axel.heidenreich@uk-koeln.de

Work +49 221 478 82108

Kerpener Str. 62
50937 Cologne

Publications - Axel Heidenreich

Link to PubMed

Group Members

Dr. med. Tim Nestler
Dr. med. Pia Paffenholz
Prof. Dr. med. David Pfister

Figure 1:
Seminoma cells of the tumor centre (TC) and at the tumor front (TF) adjacent to vascular invasion are microdissected for further analysis depicted in Figures 2-5
Figure 2:
Identification of testis cancer patients
Figure 3:
Laser microdissection of cancer cells in the tumor center and the invasion front
Figure 4:
RNA Isolation
Figure 5:
nanostring evaluation