gms | German Medical Science

Introduction: In cancer progression the development of distant metastases is the crucial adverse event.Focusing on colorectal cancer, the majority (70%) of patients develops metastasis in the liver. Within the BMBF-founded consortium MetastaSys the role of the mutational activation in the WNT pathway and the overexpression of components of the EGFR pathway are investigated in these metastases. In order to address the phenomenon of intermetastatic heterogeneity the established metastasis profiling was applied to several metastases of the same patient.

Material and methods: Two pilot cases including four and two liver metastases, respectively as well as correspondingnormal liver tissue were comprehensively characterized in terms of clinical annotations as well as standardized histopathological and molecular subtyping. All tumor samples were subtyped according to the specific mutational status of KRAS, NRAS, BRAf and PIK3CA as well as alterations in DNA mismatch repair enzymes and microsatellite instability. Fresh frozen samples for transcriptome analyses were characterized by an experienced pathologist (tumor cell content, amount of stroma, inflammatory infiltrate and necrosis) to assure high quality of tissue and to obtain valid data. After characterization, samples were profiled via subsequent transcriptome sequencing and proteomics profiling via Reverse-Phase-Protein-Arrays (RPPA).

Results: To access the clinical and genetical characterization of the samples a standardized data analysispipeline for processing the RNA-Seq data was established. The principle-component-analysis revealed a good separation between normal tissue and tumor samples. Interestingly, the correlation between healthy samples among different patients was higher than between the metastatic samples of the same patient. Within the EGFR pathway CCNB1, EGFR, FOXO1, MAP2K1, PLCXD2, RB1 and TP53 showed different expression values between normal samples and tumor tissue. Furthermore, relevant WNT markers such as AXIN2, Lef1 and CDX1 were within the top 25 upregulated genes discriminating between normal and metastatic samples.

Conclusion: RNA-Seq analysis allows a clear discrimination of normal and metastatic liver tissue. Indifferent metastases of the same patient heterogeneous expression patterns in the EGFR and the WNT pathway are identified with relevant implication for clinical treatment approaches. Based on these findings and the positive validation of the established methodology including innovative High-throughput profiling and comprehensive bioinformatics approaches we started analyzing a large number of metastatic patient samples within the consortium.