GENOMIC AND TRANSCRIPTOMIC ABERRATIONS DIFFERENTIATING TERATOMAS AT METASTATIC SITES FROM PRIMARY GERM CELL TUMORS OF TESTIS

Abstract

Introduction

Testicular germ cell tumors (TGCTs) are the most common malignancy occurring in young men between the ages of 20-40 years and include various histologically diverse cancer subtypes. Amongst all TGCTs, mature teratomas present the most advanced differentiation state and can lead to secondary malignant transformation into non-TGCT cancer types (such as sarcoma and adenocarcinoma) in various organs and late relapse with worse clinical outcome. Currently, there are no effective systemic therapies in the teratoma cases of unresectability, incomplete resection, or multifocal metastases and teratoma patients often demonstrate resistance to chemotherapy. Both primary and metastatic teratomas share several morphological similarities. However, they inhabit distinct microenvironments, potentially driven by different molecular alterations and transcriptomic vulnerabilities, which are currently limitedly studied.

Methods

We performed whole exome sequencing in 16 primary TGCTs and 17 matched post-chemotherapy pure metastatic teratomas collected from retroperitoneal lymph nodes undergoing orchiectomy. Most primary tumors (n=11) had mixed histological components, seven of them had dominant embryonal carcinoma component (>50%), two were 99-100% teratomas, one was a pure testicular seminoma. Recurrent cases were treated with one cycle of bleomycin, etoposide or cisplatin chemotherapy followed by surgical resection of residual tumor. Tumors removed from two different metastatic sites were analyzed for two patients. Tumor samples were compared to matching normal samples (blood) to distinguish germline and somatic genetic alterations. Bulk RNA-sequencing was performed in 13 residual teratomas removed from lymph nodes after chemotherapy (metastatic teratomas) and 12 primary testicular teratomas. Key findings were validated at spatial level by 10X Visium transcriptomics (Illumina platform) in one mixed TGCT sample, predominantly enriched in teratoma (95%).

Results

A total of 583 single nucleotide variants (SNVs) were identified across all 33 tumors analyzed, with the majority being missense alterations (82%). The most frequently mutated genes were associated with RTK/RAS signaling, with KRAS gain-of-function alterations being the predominant oncogenic event. Metastatic teratomas had median tumor mutation burden (TMB) of 0.65 and matched primary tumors had a lower median TMB of 0.47. GISTIC analysis revealed recurrent chromosomal 3p11.1 gain in metastatic site teratoma comparison to two primary TGCT datasets (internal and The Cancer Genome Atlas/TCGA cohort) (p-value = 0.0001). Five loci including chr10q26.3, 16p11.2, 19p13.2, 19q12 and 22q12 showed recurrent loss events in metastatic teratomas compared to primary tumors. In addition, gene set enrichment analysis of bulk RNA-sequencing data revealed significantly enriched, clinically relevant pathways e.g., collagen network formation, cell cycle checkpoint, T-cell immune response activation amongst others in metastatic compared to primary TGCTs (FDR/q-value < 0.01).

Conclusions

Both primary TGCT and metastatic teratomas demonstrate a very low TMB with no significant differences. Clinically relevant/druggable pathways were identified including T-cell activation and differentiation as well as several genes related to collagen modification were overexpressed in metastatic teratomas compared to primary tumors. Overall, we identified distinct genomic and transcriptomic features that may contribute to TGCT tumorigenesis and malignant transformation to teratomas.