Testicular germ cell cancer (TGCC) is initiated during early life from a totipotent embryonic germ cell, and the most frequent malignant cancer in young Caucasian males. The goal of this study is to determine the intratumor heterogeneity, and to unravel tumor progression from initiation till therapy-resistant metastasis. In this study, we have investigated 42 purified samples of four cases of nonseminoma with intrinsic resistance to chemotherapy including different histological elements, metastatic specimens and the precursor cancer stem cells (germ cell neoplasia in situ, GCNIS) using whole genome-, and targeted sequencing. Sequence data were used to reconstruct the evolution of these cancers. Intratumor molecular heterogeneity was observed and did not correspond to the supposed histological evolution of the primary tumor. Metastases after systemic treatment were derived from cancer stem cells frequently not identified in the primary cancer. The GCNIS mostly lacked the molecular marks of the primary TGCC and comprised dominant clones that had failed to progress into a manifest malignancy. A BRCA-like mutational signature was found without evidence for direct involvement of BRCA1 and BRCA2 genes. Our data strongly support the hypothesis that TGCC is initiated by whole genome duplication, followed by chromosome copy number alterations in the cancer stem cell population, and dynamic acquisition of chromosome arm 12p gain and accumulation of low numbers of somatic mutations resembling a BRCA-like mutational signature. These observations of heterogeneity at all stages of tumorigenesis should be considered when treating patients with GCNIS-only disease, or with clinically overt TGCC.

Molecular Heterogeneity and Early Metastatic Clone Selection in Testicular Germ Cell Cancer Development