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.

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.

Targeting CLDN6 in germ cell tumors by an antibody-drug-conjugate and studying therapy resistance of yolk-sac tumors to identify and screen specific therapeutic options.

BACKGROUND: Being the standard-of-care for four decades, cisplatin-based chemotherapy is highly efficient in treating germ cell tumors (GCT). However, often refractory patients present with a remaining (resistant) yolk-sac tumor (YST(-R)) component, resulting in poor prognosis due to lack of novel treatment options besides chemotherapy and surgery. The aim of this study was to identify novel targets for the treatment of YST by deciphering the molecular mechanisms of therapy resistance. Additionally, we screened the cytotoxic efficacy of a novel antibody-drug-conjugate targeting CLDN6 (CLDN6-ADC), as well as pharmacological inhibitors to target specifically YST. METHODS: Protein and mRNA levels of putative targets were measured by flow cytometry, immunohistochemical stainings, mass spectrometry of formalin-fixed paraffin-embedded tissues, phospho-kinase arrays, or qRT-PCR. Cell viability, apoptosis and cell cycle assays of GCT and non-cancerous cells were performed using XTT cell viability assays or Annexin V / propidium iodide flow cytometry, respectively. Druggable genomic alterations of YST(-R) tissues were identified by the TrueSight Oncology 500 assay. RESULTS: We demonstrated that treatment with a CLDN6-ADC enhanced apoptosis induction specifically in CLDN6+ GCT cells in comparison with non-cancerous controls. In a cell line-dependent manner, either an accumulation in the G2 / M cell cycle phase or a mitotic catastrophe was observed. Based on mutational and proteome profiling, this study identified drugs targeting the FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling pathways as promising approaches to target YST. Further, we identified factors relevant for MAPK signaling, translational initiation and RNA binding, extracellular matrix-related processes as well as oxidative stress and immune response to be involved in therapy resistance. CONCLUSIONS: In summary, this study offers a novel CLDN6-ADC to target GCT. Additionally, this study presents novel pharmacological inhibitors blocking FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling for the treatment of (refractory) YST patients. Finally, this study shed light on the mechanisms of therapy resistance in YST.

Assessing the risk to develop a growing teratoma syndrome based on molecular and epigenetic subtyping as well as novel secreted biomarkers.

In germ cell tumors (GCT), a growing teratoma during chemotherapy with decreasing tumor markers was defined as 'growing teratoma syndrome' (GTS) by Logothetis et al. in 1982. So far, its pathogenesis and specific treatment options remain elusive. We aimed at updating the GTS definition based on molecular and epigenetic features as well as identifying circulating biomarkers. We selected 50 GTS patients for clinical characterization and subsequently 12 samples were molecularly analyzed. We further included 7 longitudinal samples of 2 GTS patients. Teratomas (TER) showing no features of GTS served as controls. GTS were stratified based on growth rates into a slow (<0.5 cm/month), medium (0.5-1.5) and rapid (>1.5) group. By analyzing DNA methylation, microRNA expression and the secretome, we identified putative epigenetic and secreted biomarkers for the GTS subgroups. We found that proteins enriched in the GTS groups compared to TER were involved in proliferation, DNA replication and the cell cycle, while proteins interacting with the immune system were depleted. Additionally, GTSrapid seem to interact more strongly with the surrounding microenvironment than GTSslow. Expression of pluripotency- and yolk-sac tumor-associated genes in GTS and formation of a yolk-sac tumor or somatic-type malignancy in the longitudinal GTS samples, pointed at an additional occult non-seminomatous component after chemotherapy. Thus, updating the Logothetis GTS definition is necessary, which we propose as follows: The GTS describes a continuously growing teratoma that might harbor occult non-seminomatous components considerably reduced during therapy but outgrowing over time again.