Characterizing the mutational burden, DNA methylation landscape, and proteome of germ cell tumor-related somatic-type malignancies to identify the tissue-of-origin, mechanisms of therapy resistance, and druggable targets

Abstract

Background: Germ cell tumors (GCT) might undergo transformation into a somatic-type malignancy (STM), resulting in a cell fate switch to tumors usually found in somatic tissues, such as rhabdomyosarcomas or adenocarcinomas. STM is associated with a poor prognosis, but the molecular and epigenetic mechanisms triggering STM are still enigmatic, the tissue-of-origin is under debate and biomarkers are lacking.

Methods: To address these questions, we characterized a unique cohort of STM tissues on mutational, epigenetic and protein level using modern and high-throughput methods like TSO assays, 850k DNA methylation arrays and mass spectrometry.

Results and conclusions: For the first time, we show that based on DNA methylation and proteome data carcinoma-related STM more closely resemble yolk-sac tumors, while sarcoma-related STM resemble teratoma. STM harbor mutations in FGF signaling factors (FGF6/23, FGFR1/4) highlighting the corresponding pathway as a therapeutic target. Furthermore, STM utilize signaling pathways, like AKT, FGF, MAPK, and WNT to mediate molecular functions coping with oxidative stress, toxin transport, DNA helicase activity, apoptosis and the cell cycle. Collectively, these data might explain the high therapy resistance of STM. Finally, we identified putative novel biomarkers secreted by STM, like EFEMP1, MIF, and DNA methylation at specific CpG dinucleotides.

Fig. 1. Clinical and histological features of the STM cohort.

a Pie chart summarizing distribution of the various STM entities analyzed in this study. b Clinical parameters of the STM cohort (at diagnosis of STM) from the University Hospital Düsseldorf (Department of Urology) analyzed in this study. c Exemplary H&E stainings of each STM entity and IHC staining of typical marker proteins.

Fig. 2. Analyzing druggable mutations of STM tissues.

a, b Illustration of the tumor mutational burden (TMB; mutations/megabase) and microsatellite instability score (MSI; % unstable) (a) and the ratio of both parameters (b) in STM samples analyzed by the TSO assay. c All detected mutations in indicated STM samples. Blue dots label mutations found in all samples of a STM subgroup. MNV: multiple nucleotide variants; SNV: single nucleotide variants. d Overview of drugs targeting found amplified genes/signaling factors.

Fig. 3. Analyzing the proteome of STM tissues.

a A heatmap including hierarchical clustering and a Pearson’s correlation matrix illustrate similarities and differences in the proteome (abundance > 10⁷) between the various STM groups as well as YST and TER. By Venn diagrams, shared and unique proteins (abundance > 10⁷) were identified between the STM entities (b) and compared to YST/TER (c). 363 proteins were found in all analyzed STM entities (b, red circle). d STRING-based protein-protein-interaction prediction of proteins commonly found in STM entities, but not in TER or YST. e DAVID-based GO and KEGG screen for biological processes and functions related to the proteins found exclusively in each STM entity.

Fig. 4. Analyzing the DNA methylation profile of STM tissues.

a Distribution of DNA methylation levels (%) across all analyzed CpG dinucleotides. b A violin plots illustrates genome-wide distribution of DNA methylation levels. C A heatmap and a Pearson’s correlation matrix including hierarchical clustering illustrates and compares DNA methylation data, respectively. d Distribution of DNA hypo- (< 20%) and hypermethylated (> 80%) CpG dinucleotides across genomic regions/CpG islands. Venn diagrams comparing hyper- and hypomethylated CpG dinucleotides in adenocarcinomas (e) and rhabdomyosarcomas (f) with YST and TER. g Putative epigenetic biomarkers for adenocarcinomas and/or rhabdomyosarcomas based on the DNA methylation status of single CpG dinucleotides.

Fig. 5. Key findings of this study.

a Summary of found mutations common in each STM entity as well as of similarities of STM entities to YST/TER on DNA methylation and proteome level. b Summary of molecular and epigenetic processes commonly found in STM entities putatively mediating therapy resistance and interaction with cells of the immune system and the ECM. Parts of this figure were generated by biorender.com.